U.S. patent application number 14/020142 was filed with the patent office on 2014-02-06 for matrix and system for preserving biological specimens for qualitative and quantitative analysis.
This patent application is currently assigned to ViveBio, LLC. The applicant listed for this patent is ViveBio, LLC. Invention is credited to Abel De La Rosa, Mimi C. G. Healy, Anita Matthews McClernon, Daniel R. McClernon, Kristy S. Reece.
Application Number | 20140038172 14/020142 |
Document ID | / |
Family ID | 50025854 |
Filed Date | 2014-02-06 |
United States Patent
Application |
20140038172 |
Kind Code |
A1 |
De La Rosa; Abel ; et
al. |
February 6, 2014 |
Matrix and System for Preserving Biological Specimens for
Qualitative and Quantitative Analysis
Abstract
The present invention provides a device, system, and methods of
use comprising an absorbent hydrophobic polyolefin matrix, and
methods of use thereof, for storage, preserving, and recovering
liquid suspension of biological specimens containing analytes of
interest in a dry state. The dried biological specimens containing
analytes of interest absorbed on the polyolefin matrix are
reconstituted such as with molecular-grade water and released by
compressing the polyolefin matrix. The reconstituted biological
analytes are qualified for subsequent analysis, such as for
qualitative and quantitative analysis of viral nucleic acids, such
a viral load testing, genotyping, and sequencing. Also provided are
kits with instructions, and methods of use thereof, for storage,
preserving, and recovering biological specimens containing analytes
of interest using the compression device of the invention.
Inventors: |
De La Rosa; Abel;
(Alpharetta, GA) ; Healy; Mimi C. G.; (Athens,
GA) ; Reece; Kristy S.; (Pearland, TX) ;
McClernon; Daniel R.; (Cary, NC) ; McClernon; Anita
Matthews; (Cary, NC) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
ViveBio, LLC |
Alpharetta |
GA |
US |
|
|
Assignee: |
ViveBio, LLC
Alpharetta
GA
|
Family ID: |
50025854 |
Appl. No.: |
14/020142 |
Filed: |
September 6, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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PCT/US2013/053799 |
Aug 6, 2013 |
|
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14020142 |
|
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61680193 |
Aug 6, 2012 |
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Current U.S.
Class: |
435/5 ;
435/309.1 |
Current CPC
Class: |
A01N 1/0263 20130101;
G01N 1/30 20130101; C12N 1/04 20130101; A01N 1/0231 20130101 |
Class at
Publication: |
435/5 ;
435/309.1 |
International
Class: |
G01N 1/30 20060101
G01N001/30 |
Claims
1. A method for preserving and recovering a biological specimen
comprising: (a) providing a dried biological specimen in a device
comprising a container defining an interior space having side
walls, a bottom and an openable and sealable lid with an absorbent
three-dimensional polyolefin matrix removably mounted inside the
container, wherein the polyolefin matrix comprises a plurality of
interstices with a hydrophobic polyolefin surface and has contained
therein the dried biological specimen obtained from an evaporated
volume of at least 0.1 ml of a liquid suspension comprising a
solvent and the biological specimen absorbed and dried on the
matrix; (b) reconstituting the biological specimen on the
polyolefin matrix with a controlled volume of a reconstitution
media; and (c) removing the biological specimen and reconstitution
media from the polyolefin matrix by compressing the matrix.
2. The method of claim 1, wherein the polyolefin matrix comprises a
plurality of fibers having a substantially hydrophobic surface.
3. The method of claim 2, wherein the fibers within the polyolefin
matrix have a polyethylene surface.
4. The method of claim 2, wherein the fibers within the polyolefin
matrix comprise polypropylene coated with polyethylene.
5. The method of claim 4, wherein the polypropylene and
polyethylene are present in approximately equal amounts by
weight.
6. The method of claim 1, wherein the volume of the liquid
suspension is at least 0.5 ml.
7. The method of claim 1, wherein the volume of the liquid
suspension is at least 1.0 ml.
8. The method of claim 1, wherein the three-dimensional polyolefin
matrix is in a shape selected from the group consisting of a
cylinder, disk, cube, sphere, pyramid, and cone.
9. The method of claim 1, wherein the biological specimen and
reconstitution media are removed from the polyolefin matrix by
compressing the matrix in a syringe barrel.
10. The method claim 9, wherein the polyolefin matrix is compressed
by at least 50% of the volume of the polyolefin matrix.
11. The method of claim 9, wherein the polyolefin matrix is
compressed by at least 80% of the volume of the polyolefin
matrix.
12. The method of claim 1, wherein the biological specimen contains
an analyte of interest selected from the group consisting of
nucleic acids, proteins, carbohydrates, lipids, whole cells,
cellular fragments, whole virus and viral fragments.
13. The method of claim 1, wherein the biological specimen contains
an analyte of interest selected from the group consisting of DNA
and RNA.
14. The method of claim 1, wherein the biological specimen is
selected from the group consisting of whole blood, plasma, serum,
lymph, synovial fluid, urine, saliva, sputum, semen, vaginal
lavage, bone marrow, cerebrospinal cord fluid, physiological body
liquids, pathological body liquids, and combinations thereof.
15. The method of claim 1, wherein said liquid suspension
comprising said biological specimen further comprises cell
suspensions, liquid extracts, tissue homogenates, media from DNA or
RNA synthesis, saline and combinations thereof.
16. The method of claim 1, wherein the biological specimen is used
for viral load quantitation, genotyping, drug resistance testing,
or other analysis of a viral nucleic acid of interest.
17. The method of claim 16, wherein said viral nucleic acid of
interest is selected from the group consisting of HCV, HIV, HBV,
single- or double-stranded RNA viruses, single- or double-stranded
DNA viruses, retrovirus, influenza, and Parvovirus B19.
18. The method of claim 16, wherein said viral nucleic acid of
interest is contained within a genome of HCV, HIV, HBV, single- or
double-stranded RNA viruses, single- or double-stranded DNA
viruses, retrovirus, influenza, or Parvovirus B19.
19. A device for preserving and recovering a biological specimen
comprising: (a) an enclosed container defining an interior space
having side walls, a bottom and an openable and sealable lid, and
(b) an absorbent three-dimensional polyolefin matrix removably
mounted inside the container wherein the absorbent polyolefin
matrix comprises a plurality of interstices defined by a plurality
of fibers having hydrophobic polyethylene surfaces, and wherein the
absorbent polyolefin matrix can entrain a volume of at least 0.5 ml
of a liquid suspension comprising a solvent and a biological
specimen.
20. The device of claim 19, wherein the fibers of the polyolefin
absorbent matrix comprise a polypropylene core substantially coated
with a polyethylene surface.
21. The device of claim 19, wherein the polyolefin matrix is in a
shape selected from the group consisting of a cylinder, disk, cube,
sphere, pyramid, and cone.
22. The device of claim 19, wherein the polyolefin matrix can
entrain a volume of at least 1.0 ml of a liquid suspension.
23. The device of claim 19, wherein the polyolefin matrix has a
density of about 0.077 g/cc.
24. The device of claim 19, wherein the polyolefin matrix is
compressed by applying force to the plunger against the polyolefin
matrix.
25. The device of claim 24, wherein the polyolefin matrix has a
volume compressible by at least 50%.
26. The device of claim 19, wherein said biological specimen
contains RNA or DNA.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of PCT Application No.
PCT/US2013/053799 filed Aug. 6, 2013 which claims priority to U.S.
Provisional Application No. 61/680,193 filed Aug. 6, 2012, the
entire contents of which are incorporated by reference
herewith.
FIELD OF THE INVENTION
[0002] This invention relates generally to biological specimen
preserving matrix and system, devices, and methods for use
therewith. More specifically, the invention relates to a matrix and
system for collection, storage and recovery of nucleic acids such
as viral DNA and RNA specimens for subsequent quantitative and
qualitative laboratory analysis such as viral load, genotyping, and
antiviral drug resistance testing.
BACKGROUND OF THE INVENTION
[0003] Biological specimens are often collected, transported and
stored for analysis of the levels and concentrations of various
analytes contained therewithin. Conventionally, liquid suspensions
of biological specimens are stored in sealed airtight tubes under
refrigeration. Liquid sample collection, handling, transportation
and storage has many problems associated with it, for example: the
cost of refrigeration (typically by dry ice) in remote collection
centers; the risk of container breakage or leakage which causes
loss of sample and the danger of infection; sample instability
during shipment and storage; refusal of transport carriers to
accept liquid biohazard shipments; and collection of adequate
sample volume to ensure quantities compatible with laboratory
methods of subsequent qualitative and quantitative analyses. The
costs of addressing the above problems are substantial.
[0004] Dried blood spot (DBS) and dried plasma spot (DPS) sampling
on filter paper are alternative methods to the liquid sampling
procedures, and have been used worldwide with some success. Since
the 1980s, manufacturers such as Schleicher and Schuell Corp.,
Bio-Rad, Boehringer Mannheim Corp., and Whatman, Inc., have been
producing filter papers for DBS and DPS sampling. In using these
commercially available biological sampling filter paper systems, a
blood or plasma spot is placed in one or more designated areas of
the filter paper, allowed to dry, and then mailed along with a test
request form to the laboratory. Commonly used filter papers are
known to those of ordinary skill in the art, such as Whatman 3 MM,
GF/CM30, GF/QA30, S&S 903, GB002, GB003, or GB004. Several
categories of blotting materials for blood specimen collection are
available, e.g., S&S 903 cellulose (wood or cotton derived)
filter paper and Whatman glass fiber filter paper. However, certain
disadvantages have been associated with these commercially
available filter papers. Specifically, certain of these
commercially available and commonly used materials lack
characteristics which provide precision values and accuracy that
are preferred for carrying out certain qualitative and quantitative
biological assays.
[0005] Genetic material can be extracted and isolated from prior
art DBSs in sufficient quantities for use in genetic analysis. For
instance, DBS has been used for the detection of prenatal human
immunodeficiency virus (HIV) infection by the polymerase chain
reaction (PCR) (Cassol, et al., J. Clin Microbiol. 30 (12):
3039-42, 1992). DPS and DBS have also been used with limited
success for HIV RNA detection and quantification (Cassol, et al.,
J. Clin. Microbiol. 35: 2795-2801, 1997; Fiscus, et al., J. Clin.
Microbiol. 36: 258-60, 1998; O'Shea, et al., AIDS 13: 630-1, 1999;
Biggar, et al., J. Infec. Dis. 180 1838-43, 1999; Brambilla, et
al., J. Clin. Microbiol. 41(5): 1888-93, 2003); HIV DNA detection
and quantification (Panteleefe, et al., J. Clin. Microbiol. 37:
350-3, 1999; Nyambi, et al., J. Clin. Microbiol. 32: 2858-60,
1994); and HIV antibody detection (Evengard, et al., AIDS 3: 591-5,
1989; Gwinn, et al., JAMA 265: 1704-08, 1991). HCV RNA detection
and genotyping are also reported using DBS (Solmone et al., J.
Clin. Microbio. 40 (9): 3512-14, 2002). Although these studies
provide a good correlation with titers using DPS or DBS is obtained
as compared with conventional liquid plasma samples, a loss of
viral titers may occur after room temperature storage (Cassol, et
al., J. Clin. Microbiol. 35: 2795-2801, 1997; Fiscus, et al., J.
Clin. Microbiol. 36: 258-60, 1998). DBS and DPS samples are clearly
less expensive and less hazardous to transport than liquid
samples.
[0006] However, the procedure of analyte microextraction from DBS
and DPS on filter paper suffers from a number of disadvantages. For
example, microextraction of sufficient DNA or RNA from filter paper
involves reconstitution in a liquid medium under certain vigorous
procedures, e.g., vortex and centrifugation that damages the
genetic analytes of interest. Furthermore, the fibers and other
components of the filters become dislodged into the reconstitution
solution, and require further centrifugation separation and/or can
impede the ability to isolate the genetic material, such as by
blocking genetic material from adhering to a separation column.
Such prior microextraction procedures require a high standard of
technical assistance, and even then do not consistently provide
results with a desired level of sensitivity, reproducibility,
quantification and specificity.
[0007] Furthermore, the sample volume used for DBS and DPS on
filter paper is limited, typically to 50-200 ul spots, and
considerable difficulty in analyte detection and accurate
quantification and reproducibility can be encountered, particularly
when the concentration of the desired analyte material is low in
the sample. Also in the prior art, there is a lack of deliberate
inhibition of enzymes and chemicals which degrade the analytes,
such as genetic material contained therewithin. Even in the
presence of a bacteriostatic agent there are conditions that permit
enzymatic, nonenzymatic and autolytic breakdown of the genetic
material. Furthermore, microextraction of genetic material from DBS
or DPS on filter papers is considerably more difficult if
absorption of high molecular weight DNA or RNA is required.
Although the introduction of new material and transportation
methods continuously improve the ways samples are handled, the
quantity and quality of the sample available for subsequent
analysis are still of great concern to researchers and clinicians
alike.
[0008] U.S. Pat. No. 7,638,099 provides an advantageous alternative
system for biological sample collection, storage and
transportation. The reference suggests the use of cellulose acetate
fibers and hydrophilic polymer fibers as being advantageous for an
absorbent matrix material. However, further improvements are
desired for certain situations, such as to achieve more accurate
and reproducible quantification of viral load in a sample.
[0009] Thus, there is a need for an improved device for collection,
storage and transportation of liquid suspension of biological
specimens containing analytes of interest in a dry state,
especially in large field studies and for application in settings
where collection, centrifugation, storage and shipment can be
difficult, as is often the case in developing countries. In
addition, there is a need for improved recovery of viral specimens
for subsequent analysis that provides precision values and accuracy
of detection, reproducibility and quantification of the analytes of
interest contained therewithin.
SUMMARY OF THE INVENTION
[0010] This invention fulfills in part the need to provide a safe,
convenient and simple device and method for preserving, storage and
transportation of biological specimens containing analytes of
interest. The invention also fulfills in part the need to recover
biological specimens containing analytes of interest for subsequent
analysis that provides more desirable sensitivity and specificity
of detection. More particularly, the invention provides an improved
matrix storage material comprising hydrophobic polyolefin polymers
for use as a device, system, and method for accurate and
reproducible quantification of viral load in a patient. The
invention provides a novel device and method for preserving,
storing, and transporting a liquid suspension of biological
specimens in a dry state and further reconstituting the analytes of
interest contained in the biological specimens for use in research
and site validated clinical testing.
[0011] In certain embodiments, the absorbent polyolefin matrix
comprises hydrophobic polymers, including polyethylene. In certain
embodiments, the absorbent polyolefin fiber matrix comprises a
hydrophobic polyethylene surface coating. In certain embodiments,
the matrix comprises a plurality of polyolefin fiber strands,
wherein each individual fiber strand within the absorbent
polyolefin fiber matrix is composed of a core and an outer sheath.
In certain embodiments, the core of each fiber comprises
polypropylene, and the outer coating sheath of each fiber comprises
polyethylene. In certain embodiments, each individual fiber strand
within the polyolefin fiber matrix is composed of a core of each
strand comprising about 50% polypropylene and a hydrophobic outer
sheath surrounding the core of each strand comprising about 50%
polyethylene.
[0012] Based on the data presented, the invention provides that a
hydrophobic polyolefin fiber matrix is superior compared to
previous dried collection devices for absorption, preservation,
stabilisation, and subsequent recovery of nucleic acid for
quantification and qualification. Without wishing to be bound by
theory, it is believed that these surprising results are due to the
properties of the embedded hydrophobic interstices, or pockets
within the polyolefin matrix. These pockets provide a reservoir for
the analyte to reside while excluding water from the analyte, e.g.,
nucleic acid, providing a stable environment during storage. The
improved hydrophobic polyolefin matrix further allows polar
solvents to evaporate more consistently and efficiently. Therefore,
the improved polyolefin matrix retains analytes and suspended
particles inside the matrix better than for example a cellulose
matrix. Contrary to the teachings in the prior art that hydrophilic
polymer surfaces in the matrix are more desirable, it has been
discovered that hydrophobic polyolefin surfaces in the matrix are
surprisingly advantageous. Thus, a substantially intact viral
nucleic acid, for example, can be eluted from the reconstituted
matrix with great efficiency permitting a surprisingly accurate
degree of quantification and qualification of the viral load in the
biological sample.
[0013] The polyolefin fiber matrix of the invention absorbs greater
than 0.05 ml of a liquid suspension of biological specimens
absorbed and dried thereon. In certain embodiments, the polyolefin
fiber matrix absorbs at least 0.1, ml or 0.5, ml of the liquid
suspension. In yet other embodiments, the polyolefin fiber matrix
absorbs at least 1 ml, 1.5 ml, 2.0 ml, 2.5 ml, 3.0 ml, or more, of
the liquid suspension of biological specimens.
[0014] The invention provides that the absorbent polyolefin fiber
matrix, while harboring numerous hydrophobic pockets, is able to be
compressed by applying force against the matrix by at least 10% of
the volume of the matrix to release a portion of the re-suspended
biological specimen stored therewithin. In other embodiments, the
matrix is able to be compressed by at least 20%, 50%, 75%, 80%,
85%, 90%, or 95% or more of the volume of the matrix to release a
portion of the liquid suspension of biological specimen stored in
the matrix. In other words, the matrix is at least 10% porous or
defines at least 10% available space, including numerous
hydrophobic pockets within the polyolefin fiber matrix, for the
storage of a biological specimen therein.
[0015] In certain embodiments, the polyolefin fiber matrix is
three-dimensional in a variety of different shapes, including but
not limited to, a cylinder, disk, cube, sphere, pyramid, cone,
concave, indented, invaginated or other shapes and surface textures
suitable for absorption and fitting inside a container. In certain
embodiments, the matrix is in the shape of a cylinder about 18 mm
to 24 mm, or 21 mm, in length, and 5 mm to 15 mm, or 9 mm, in
diameter, with a density of about 0.01 g/cc to 0.1 g/cc, or about
0.077 g/cc. In certain embodiments, a majority of the polyolefin
fiber sizes are in the range of about 1-100 microns, 10-50 microns,
or 20-25 microns and contain numerous hydrophobic pockets.
[0016] The invention provides a device and methods that allow for
biological testing of air-dried bodily fluid samples without the
need for refrigerated or frozen shipping and storage. The inventive
device and methods provide the capability to significantly reduce
the costs of shipping infectious materials worldwide, especially
those associated with large clinical trials. Moreover, the
inventive device and methods for preserving biological specimens
are applicable to and include a wide range of esoteric and standard
clinical testing, including qualitative and quantitative nucleic
acid analysis.
[0017] In certain embodiments, the invention provides a device, and
method of use thereof, for preserving and recovering a biological
specimen containing analytes of interest. More particularly, the
device comprises a first enclosed container defining an interior
space having side walls, a bottom and an openable and sealable lid
or cap. In certain embodiments, the first enclosed container is a
tube having a sealable cap with an absorbent three-dimensional
polyolefin fiber matrix mounted therein. In certain embodiments, an
interior of the tube or cap has an internal surface extension with
the absorbent three-dimensional matrix removably mounted
thereon.
[0018] In certain embodiments, the invention further comprises a
second enclosed compression container with a syringe barrel shape
or any other suitable shape for receiving therein the matrix for
reconstitution, compression, and release of the analytes of
interest, e.g., intact viral RNA or DNA. In certain embodiments,
only one container is required for storage, transportation,
reconstitution, and release of the analytes of interest from the
matrix.
[0019] In certain embodiments, the device may optionally comprise a
desiccant inside the enclosed container in vaporous communication
with the matrix to maintain a dried state of the matrix and
integrity of the biological specimen and analytes of interest it
contains on the matrix. Exemplary suitable desiccant includes, but
is not limited to, montmorillonite clay, lithium chloride,
activated alumina, alkali alumino-silicate, DQ11 Briquettes, silica
gel, molecular sieve, calcium sulfate, or calcium oxide. In certain
embodiments, the desiccant indicates its moisture content by
colorimetric means. In other embodiments, since unlike the
hydrophilic cellulose acetate matrix where the solvents are not
released as efficiently, the hydrophobic polyolefin fiber matrix of
the invention allows the solvents to evaporate more consistently
and efficiently, and a desiccant is not necessary.
[0020] According to the invention, the analytes of interest
include, but are not limited to, nucleic acids, proteins,
carbohydrates, lipids, whole cells, cellular fragments, whole virus
or viral fragments. In certain embodiments, the analytes of
interest are nucleic acids including either or both DNA and RNA
molecules. The invention particularly provides improved systems and
methods for the detection and quantitation of RNA, e.g., whole
virus for determining viral load and genotyping in a biological
specimen or subject.
[0021] In certain embodiments, the nucleic acid of interest is HCV
or other single stranded RNA viruses. In certain embodiments, the
nucleic acid of interest is HIV or other retroviruses. In certain
embodiments, the nucleic acid of interest is HBV or other double
stranded DNA viruses. In certain embodiments, the nucleic acid of
interest is Influenza or other double stranded RNA viruses. In
certain embodiments, the nucleic acid of interest is Parvovirus B19
or other single stranded DNA viruses. In certain embodiments, the
nucleic acid of interest is contained within the HCV genome or the
genome of other single stranded RNA viruses. In certain
embodiments, the nucleic acid of interest is contained within the
HIV genome or the genome of other retrovirus. In certain
embodiments, the nucleic acid of interest is HBV genome or the
genome of other double stranded DNA viruses. In certain
embodiments, the nucleic acid of interest is Influenza genome or
the genome of other double stranded RNA viruses. In certain
embodiments, the nucleic acid of interest is Parvovirus B19 or the
genome of other single stranded DNA virus.
[0022] According to the invention, the biological specimens
include, but are not limited to, whole blood, plasma, urine,
saliva, sputum, semen, vaginal lavage, bone marrow, cerebrospinal
fluid, other physiological or pathological body liquids, or any of
the combinations thereof. In certain embodiments, the biological
specimen is human body fluid, such as whole blood containing the
analytes of interest, such as nucleic acids, including either or
both DNA and RNA molecules. In certain embodiments, the analytes of
interest are nucleic acids and the biological specimens comprise at
least 5 ng to 1 .mu.g either or both DNA or RNA molecules. In yet
other embodiments, the biological specimen is contained in liquid
suspension. According to the invention, the liquid suspension
includes, but is not limited to, cell suspension, liquid extracts,
tissue homogenates, media from DNA or RNA synthesis, saline, or any
combinations thereof.
[0023] The invention further provides a system and method for
preserving and recovering a biological specimen containing analytes
of interest, such as RNA, from the matrix in the device provided by
the invention. In certain embodiments, the method comprises the
following steps of providing a device comprising an absorbent
matrix comprised of hydrophobic polyolefin fibers, wherein in
certain embodiments each strand of fiber is comprised of a core and
an outer sheath surface, wherein said core of each strand comprises
polypropylene, and said outer sheath surface of each strand
comprises polyethylene. In the method the matrix can be provided
with a dried biological specimen contained thereon obtained from a
volume of at least 0.05 ml of an evaporated liquid suspension
comprising a liquid and the biological specimen containing analytes
of interest. The method further comprises reconstituting the
biological specimen on the matrix with a controlled volume of a
reconstitution medium; and removing the biological specimen from
the matrix, such as by compressing the matrix.
[0024] In certain embodiments, the reconstitution solution is water
medium. In other embodiments, the reconstitution buffer comprises
1.times. phosphate buffered saline (PBS) or nuclease-free water
optionally comprising sodium azide or other antimicrobial agent. In
yet other embodiments, the reconstitution buffer is a "lysis"
buffer. The reconstitution buffer may also include any number or
combinations of available biological preservatives or blood
anticoagulants including, but not limited to,
ethylenediaminetetraacetic acid (EDTA), sodium citrate, and
heparin.
[0025] In one embodiment, the method can comprise removing the
matrix from the container prior to compressing the matrix in a
second container, e.g., a syringe barrel. In yet another
embodiment, the compression of the matrix is achieved by applying
force against the hydrophobic polyolefin matrix within the same
container to release the analytes of interest. According to the
invention, the hydrophobic polyolefin matrix in the compression
device is capable of compressing by at least 10%, 20%, 25%, 50%,
55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or more of the volume of the
matrix to release a portion of the biological specimen suspended in
the matrix.
[0026] The invention further provides a kit for preserving a liquid
suspension of a biological specimen containing analytes of interest
and for follow-up recovery and analysis. In certain embodiments,
the kit includes the compression device provided by the present
invention and instructions for preserving the biological specimens
containing analytes of interest. The kit can further comprise a
stabilising solution to inhibit degradation of the analytes. The
kit can further comprise a reconstitution medium, a compression
device and further instructions for recovery the analytes of
interest contained in the biological specimen. In certain
embodiments, the compression device comprises a tube with a syringe
barrel shape that contains a plunger with a cup attached that the
matrix is permanently adhered to allowing compression of the matrix
to be achieved by applying force to the plunger, and wherein at
least 10% to 90%, or greater, of the volume of the matrix is
compressed to release a portion of the bound biological
specimen.
[0027] The invention further provides subsequent analysis using the
recovered biological specimen containing analytes of interest. In
certain embodiments, the analytes of interest are RNA molecules
that are detected or analyzed using analytical and diagnostic
methods known in the art. In certain embodiments, the analytes of
interest are intact virus, such as HCV or HIV, and the biological
specimen recovered from the device is used for evaluation and
analytical measurements with reproducibility, accuracy, and
precision.
BRIEF DESCRIPTION OF THE DRAWINGS
[0028] FIG. 1A is a perspective view of an assembled device
according to one embodiment of the invention. FIG. 1B is a
perspective view of a disassembled device according to one
embodiment of the invention ready for sample addition.
[0029] FIG. 2 illustrates addition of sample to the polyolefin
matrix of a device according to one embodiment of the
invention.
[0030] FIG. 3 illustrates addition of sample to the polyolefin
matrix of a device according to one embodiment of the
invention.
[0031] FIG. 4 is a perspective view of preparing to transfer the
polyolefin matrix of a device according to one embodiment of the
invention into an empty syringe barrel.
[0032] FIG. 5 is a perspective view of completed delivery of the
polyolefin matrix into the syringe barrel.
[0033] FIG. 6 illustrates rehydration of the polyolefin matrix by a
pipette tip gently placed on the top of the matrix and slowly
dispensing reconstitution buffer.
[0034] FIG. 7A illustrates insertion of the plunger into the
syringe barrel. FIG. 7B illustrates application of pressure to the
syringe plunger. FIG. 7C illustrates compression of the polyolefin
matrix plug. FIG. 7D illustrates completion of sample recovery.
[0035] FIG. 8 provides a linear regression analysis for matrix
comparison studies using the Abbott REALTIME HBV assay.
[0036] FIG. 9 provides a sample correlation and HCV viral load
using fresh plasma and plasma samples processed through the ViveST
devices of the invention.
[0037] FIG. 10 provides a sample correlation and HIV-1 viral load
using fresh plasma and plasma samples processed through the ViveST
devices of the invention and recovered with mLysis.
[0038] FIG. 11 provides a sample correlation and HIV-1 viral load
using fresh plasma and plasma samples processed through the ViveST
devices of the invention and recovered with water.
[0039] FIG. 12 provides a HCV analytical measurement range
determination using the Abbott REALTIME HCV assay for samples
processed through the ViveST devices of the invention.
[0040] FIG. 13 provides comparisons of frozen plasma and plasma
samples processed through the ViveST devices of the invention using
the Roche COBAS AmpliPrep/COBAS TaqMan HCV assay.
[0041] FIG. 14 provides comparisons of frozen plasma and plasma
samples processed through the VivesST devices of the invention
using the Roche COBAS AmpliPrep/COBAS TaqMan HIV assay.
[0042] FIG. 15 provides a HIV-1 analytical measurement range
determination using the Abbott REALTIME HIV-1 assay for samples
processed through the ViveST devices of the invention.
[0043] FIG. 16 provides a linear regression analysis of HCV 7-day
stability studies at ambient conditions using the Abbott REALTIME
HCV assay for samples processed through the ViveST devices of the
invention.
[0044] FIG. 17 provides comparisons of target and actual HCV titers
in the 7-day stability studies at ambient conditions using the
Abbott REALTIME HCV assay, viewed by concentration level.
[0045] FIG. 18 provides comparisons of HCV titers analyzed on
initial test point (Day 1) in the nominal frozen plasma not
processed through the ViveST devices of the invention, and the HCV
titers of the plasma samples stored on the ViveST device for 7 days
and processed through the ViveST devices thereafter.
[0046] FIG. 19 provides a linear regression analysis of HCV 21-day
stability studies at ambient storage condition using the Abbott
REALTIME HCV assay for samples processed through the ViveST devices
of the invention.
[0047] FIG. 20 provides comparisons of target and actual HCV titers
in the 21-day stability studies at ambient storage condition using
the Abbott REALTIME HCV assay, view by concentration level.
[0048] FIG. 21 provides a linear regression analysis of HCV 21-day
stability studies at 4.degree. C. storage condition using the
Abbott REALTIME HCV assay for samples processed through the ViveST
devices of the invention.
[0049] FIG. 22 provides comparisons of target and actual HCV titers
in the 21-day stability studies at 4.degree. C. storage condition
using the Abbott REALTIME HCV assay, view by concentration
level.
[0050] FIG. 23 provides a linear regression analysis of HCV 21-day
stability studies at 40.degree. C./75% RH storage condition using
Abbott REALTIME HCV assay for samples processed through the ViveST
devices of the invention.
[0051] FIG. 24 provides comparisons of target and actual HCV titers
in the 21-day stability studies at 40.degree. C./75% RH storage
condition using the Abbott REALTIME HCV assay, view by
concentration level.
[0052] FIG. 25 provides comparisons of target and actual HCV titers
in the 21-day stability studies, view by storage condition after 21
days storage.
[0053] FIG. 26 provides a linear regression analysis of HIV-1
stability studies at ambient conditions using the Abbott REALTIME
HIV-1 assay for samples processed through the ViveST devices of the
invention.
[0054] FIG. 27 provides comparisons of target and actual HIV-1
titers in the HIV-1 stability studies at ambient conditions using
the Abbott REALTIME HIV-1 assay, view by concentration level.
[0055] FIG. 28 provides a linear regression analysis of HCV 62-day
stability studies at ambient storage conditions using the Abbott
REALTIME HCV assay for samples processed through the ViveST devices
of the invention.
[0056] FIG. 29 provides comparisons of target and actual HCV titers
in the 62-day stability studies at ambient storage condition using
the Abbott REALTIME HCV assay, view by concentration level.
[0057] FIG. 30 provides a linear regression analysis of HCV 62-day
stability studies at 4.degree. C. storage condition using the
Abbott REALTIME HCV assay for samples processed through the ViveST
devices of the invention.
[0058] FIG. 31 provides comparisons of target and actual HCV titers
in the 62-day stability studies at 4.degree. C. storage condition
using the Abbott REALTIME HCV assay, view by concentration
level.
[0059] FIG. 32 provides a linear regression analysis of HCV 62-day
stability studies at 40.degree. C./75% RH storage conditions using
the Abbott REALTIME HCV assay for samples processed through the
ViveST devices of the invention.
[0060] FIG. 33 provides comparisons of target and actual HCV titers
in the 62-day stability studies at 40.degree. C./75% RH storage
conditions using the Abbott REALTIME HCV assay, view by
concentration level.
[0061] FIG. 34 provides comparisons of target and actual HCV titers
in the 62-day stability studies, view by storage condition after 62
days storage.
[0062] FIG. 35 provides a linear regression analysis of HIV-1
62-day stability studies at ambient storage conditions using the
Abbott REALTIME HIV-1 assay for samples processed through the
ViveST devices of the invention.
[0063] FIG. 36 provides comparisons of target and actual HIV-1
titers in the 62-day stability studies at ambient storage
conditions using the Abbott REALTIME HIV-1 assay, view by
concentration level.
[0064] FIG. 37 provides a linear regression analysis of the HIV-1
62-day stability studies at 4.degree. C. storage condition using
the Abbott REALTIME HIV-1 assay for samples processed through the
ViveST devices of the invention.
[0065] FIG. 38 provides comparisons of target and actual HIV-1
titers in the 62-day stability studies at 4.degree. C. storage
conditions using the Abbott REALTIME HIV-1 assay, view by
concentration level.
[0066] FIG. 39 provides a linear regression analysis of HIV-1
62-day stability studies a 40.degree. C./75% RH storage conditions
using the Abbott REALTIME HIV-1 assay for samples processed through
the ViveST devices of the invention.
[0067] FIG. 40 provides comparisons of target and actual HIV-1
titers in the 62-day stability studies at 40.degree. C./75% RH
storage conditions using the Abbott REALTIME HIV-1 assay, view by
concentration level.
[0068] FIG. 41 provides comparisons of target and actual HIV-1
titers in the 62-day stability studies, view by storage condition
after 62 days storage.
[0069] FIG. 42 provides a linear regression analysis for target and
achieved frozen plasma samples.
[0070] FIG. 43 provides a probit analysis for HIV-1 limited of
detection (LOD) evaluation.
[0071] FIG. 44 provides a regression analysis for samples processed
through the ViveST device of the invention and the frozen plasma
using the Roche COBAS TaqMan HCV Test (v2.0).
[0072] FIG. 45 provides a linear regression analysis of HCV 7-day
stability studies at ambient storage conditions using the Roche
COBAS TaqMan HCV Test (v 2.0) for samples processed through the
ViveST devices of the invention.
[0073] FIG. 46 provides comparisons of target and actual HCV titers
in the 7-day stability studies at ambient storage conditions using
the Roche COBAS TaqMan HCV Test (v 2.0).
[0074] FIG. 47 provides a linear regression analysis for target and
achieved frozen plasma samples.
[0075] FIG. 48 provides a probit analysis for HCV LOD/LOQ
Studies.
[0076] FIG. 49 illustrates 1.0 mL loaded on 3 matrixes. Picture
taken at time of loading demonstrating that all matrixes completely
absorbed all material.
[0077] FIG. 50 illustrates 1.0 mL, 1.5 mL, and 2.0 mL loaded.
Picture taken at time of loading. With 1.5 mL it was observed that
specimen was not completely absorbed and liquid pooled in the inner
rim of the inverted cap. With 2.0 mL it was observed that specimen
was not completely absorbed and liquid flowed over the inner rim of
the inverted cap and pooled in the outer rim of the inverted
cap.
[0078] FIG. 51 illustrates 1.0 mL, 1.5 mL, and 2.0 mL loaded.
Picture taken 30 minutes after loading. With 1.5 mL it was observed
that all specimens were completely absorbed by the matrix. With 2.0
mL it was observed that specimen was not yet completely absorbed
and liquid still pooled in the outer rim of the inverted cap.
[0079] FIG. 52 illustrates 1.0 mL, 1.5 mL, and 2.0 mL loaded.
Picture taken after drying overnight. All matrixes were dried. With
1.5 mL matrix it was observed that all specimens were completely
absorbed by the matrix and dried. With 2.0 mL it was observed that
the matrix did not absorb all specimens and dried specimen was
visible in the outer rim of the inverted cap.
[0080] FIG. 53 provides a box plot of results of HIV-1
concentration study using the Abbott REALTIME HIV-1 Assay.
[0081] FIG. 54 provides a scatter plot of Samples (n=180) processed
through the ViveST device of the invention using the Abbott
REALTIME HCV Assay.
[0082] FIG. 55 provides a regression analysis for samples processed
through the ViveST devices of the invention and the frozen plasma
sample using the Abbott REALTIME HBV Assay.
[0083] FIG. 56 provides a linear regression analysis of HBV 60-day
stability studies at ambient storage conditions using the Abbott
REALTIME HBV assay for samples processed through the ViveST device
of the invention.
[0084] FIG. 57 provides comparisons of target and actual titers in
the 60-day stability studies at ambient storage conditions using
the Abbott REALTIME HBV Assay.
[0085] FIG. 58 provides a linear regression analysis for target and
achieved frozen plasma samples.
[0086] FIG. 59 provides a probit analysis for HBV LOD/LOQ
studies.
DETAILED DESCRIPTION OF THE INVENTION
[0087] The invention may be understood more readily by reference to
the following detailed description of the preferred embodiments of
the invention and the Examples included herein. However, before the
present devices, materials, and methods are disclosed and
described, it is to be understood that this invention is not
limited to specific embodiments of the devices, materials and
methods, as such may, of course, vary, and the numerous
modifications and variations therein will be apparent to those
skilled in the art. It is also to be understood that the
terminology used herein is for the purpose of describing specific
embodiments only and is not intended to be limiting.
[0088] The invention provides a device and method for collection,
storage and transportation of a liquid suspension of a biological
specimen containing an analyte of interest. More particularly, the
present invention provides a device and method for collection,
storage and transportation of a liquid suspension containing a
biological specimen in a dry state that is convenient and simple to
use. As used herein, the terms "a" or "an" mean one or more than
one depending upon the context in which they are used. For example,
"an analyte" in a sample refers to a particular type of analyte of
interest (e.g., such as intact HCV or HIV RNA), of which there may
be numerous copies within the sample. Where a sample is referred to
as containing an analyte, it is understood that the sample may
contain many other types of analytes of interest also.
[0089] According to the invention, the time period for which
biological specimen may be preserved may be as short as the time
necessary to transfer a sample of biological specimen from a
collection source to the place where subsequent analysis is to be
performed. Therefore, the invention provides that such preservation
can occur for a period of several minutes, hours, days, months, or
greater. The temperature conditions under which a biological
specimen may be stored in the device provided by the invention are
not limited. Typically, samples are shipped and/or stored at
ambient or room temperature, for example, from about 15.degree. C.
to about 40.degree. C., preferably about 15.degree. C. to
25.degree. C. In another embodiment the samples may be stored in a
cool environment. For example, in short-term storage, the samples
can be refrigerated at about 2.degree. C. to about 10.degree. C. In
yet another example, the samples may be refrigerated at about
4.degree. C. to about 8.degree. C. In another example, in long-term
storage, the samples can be frozen at about -80.degree. C. to about
-10.degree. C. In yet another example, the samples can be frozen
from about -60.degree. C. to about -20.degree. C. In addition, the
device may preferably but not necessarily be stored in dry or
desiccated conditions or under an inert atmosphere.
[0090] In certain embodiments, the invention provides a device
comprising a first enclosed container defining an interior space
having side walls, a bottom and an openable and sealable lid or cap
with an absorbent three-dimensional hydrophobic polyolefin fiber
matrix disposed inside the first enclosed container. The invention
can further comprise a second container with a syringe barrel-shape
or any other suitable shape, and a plunger contained therewith,
wherein the matrix can be placed therein for compression and
release of the analyte of interest. In certain embodiments, the
matrix can be loaded with a biological specimen and dried, and
placed into a single container which serves both a protective, dry
transportation vessel and is configured for compression of the
reconstituted matrix for release of the analyte of interest.
[0091] The shape of the first or second container is not limited,
but can be cylindrical, rectangular, or tubular for example.
Materials for construction of the containers are not limited, but
can be plastic, metal foil, laminate comprising metal foil,
metallized film, glass, silicon oxide coated films, aluminum oxide
coated films, liquid crystal polymer layers, and layers of
nano-composites, metal or metal alloys, acrylic, and amorphous
carbon for example. In certain embodiments, the invention provides
a first enclosed container having a threaded screw cap. In other
embodiments, the lid or cap can remain attached to the first
enclosed container such as a flip-top fashion. In yet other
embodiments, the lid or cap may also be cork-like or any other
openable configuration. The lid or cap can also provide an
air-tight seal when the first enclosed container is closed.
[0092] The device also comprises a hydrophobic polyolefin fiber
matrix for retaining the biological specimen, drying the analyte of
interest therein, reconstitution and release of the analyte. In
certain embodiments, the hydrophobic matrix is made from polyolefin
fibers that can be quality-controlled during manufacturing. As used
herein, the term "polyolefin fiber matrix" refers to a fiber matrix
made of at least one type polyolefin polymer produced from a simple
olefin (also called an alkene with the general formula
C.sub.nH.sub.2n) as a monomer. The term "hydrophobic" polyolefin
surface is used to describe a polyolefin surface that generally
repels water or resists wetting, for example, as would result from
minimal or substantially absent hydrogen bonding or other chemical
bonding interactions between the polyolefin surface and water
molecules. A hydrophobic polyolefin surface generally lacks the
molecular entities or substituents to interact with the polar
solvents, in particular water, or with other polar groups. In one
aspect, the hydrophobicity of a polyolefin surface can be
quantified by the contact angle, .theta..sub.C, which is the angle
between the polyolefin surface and the tangent to the water surface
at the contact point, that is, where the water/air (or water/vapor)
interface meets the polyolefin surface. For example, the polyolefin
surface can be considered "hydrophobic" if the water contact angle
is greater than about 85.degree.. In another aspect, the polyolefin
surface can be considered "hydrophobic" if the water contact angle
is greater than about 90.degree.; alternatively, greater than about
95.degree.; alternatively, greater than about 100.degree.;
alternatively, greater than about 105.degree.; alternatively,
greater than about 110'; alternatively, greater than about 115'; or
alternatively, greater than about 120.degree..
[0093] In certain embodiments, the hydrophobic polyolefin fiber
matrix comprises fibers that have a hydrophobic first polyolefin,
such as polyethylene surface. In certain embodiments the surface
can be a coating or sheath substantially disposed on a core of a
second polyolefin, such as polypropylene. The relative amounts of
each polymer can range from 10%-90% polyethylene and 10%-90%
polypropylene, and in some embodiments about 50% polyethylene and
about 50% polypropylene by weight. The hydrophobic polymer fibers
are bound together and shaped as is known in the art and
commercially available, such as from Filtrona Porous Technologies,
with pore sizes ranging from 2 microns to 100 microns.
[0094] In certain embodiments, the hydrophobic polyolefin matrix of
the invention is an absorbent material to which the liquid
suspension of biological specimen containing analytes of interest
will be retained and which does not inhibit evaporation of the
solvent (e.g., water or other fluids) for storage or subsequent
reconstitution and analysis of the analytes of interest applied
thereto. The matrix of the invention comprises hydrophobic
polyolefin surfaces of a porous nature to provide entrainment of
the liquid suspension in the matrix. As used herein, the term
"entrain" and derivatives thereof means that the liquid suspension
of a polar solvent and analytes can be temporarily entrapped within
the interstices, or pores, of the matrix without substantial
reliance on chemical and/or physical interactions such that a polar
solvent like water can evaporate and leave the suspended analytes
remaining in the matrix.
[0095] A matrix suitable for this purpose includes, but is not
limited to, a matrix that comprises or is composed of hydrophobic
polyolefin homopolymers and copolymers. Particularly suited are
polymers of ethylene alone, combined or copolymerized with an
alpha-olefin polymer. Examples of the alpha-olefin polymer include,
but are not limited to, propylene, 1-butene, 2-butene,
3-methyl-1-butene, isobutylene, 1-pentene, 2-pentene,
3-methyl-1-pentene, 4-methyl-1-pentene, 1-hexene, 2-hexene,
3-hexene, 3-ethyl-1-hexene, 1-heptene, 2-heptene, 3-heptene, the
four normal octenes, the four normal nonenes, or the five normal
decenes. In another aspect, the alpha-olefin polymer may be
selected from 1-butene, 1-pentene, 1-hexene, 1-octene, 1-decene, or
styrene. In certain embodiments, the hydrophilic olefin polymers
form the core, and hydrophobic polymers such as made with
polyethylene form the outer sheath surface of each strand of the
polyolefin fiber matrix of the invention.
[0096] Any ratio of polymers can be employed to prepare the
suitable polyolefin polymer matrix for use herein. For example,
ethylene can be used from about 5 to about 95 mole percent for the
outer sheath surface of each strand, and any of the suitable
monomers can constitute the balance of the mole percent of the
alpha olefins for the core of each strand. Thus, ethylene can be
used from about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65,
70, 75, 80, 85, 90, or 95 mole percent to prepare suitable
material, with any of the suitable monomers can constitute the
balance of the mole percent of the alpha olefins. Alternatively,
ethylene can be used from about 5 to about 95 mole percent, about
15 to about 85 mole percent, or about 25 to about 75, about 35 to
about 65, or about 45 to about 55 mole percent, with any of the
suitable monomers making up the balance of the mole percent of the
alpha olefins. In certain embodiments, polyethylene is used for the
outer sheath surface, and polypropylene is used for the core, of
each strand within the polyolefin fiber matrix of the invention.
Polyolefin polymers can be low density or high density, highly
branched or substantially unbranched, and the like, as long as the
polymer can withstand the methods used to prepare and use the
disclosed devices and methods. In certain embodiments, the density
of the resulting polyolefin fiber matrix of the invention is about
0.077 grams/cc.
[0097] Thus, the polyolefin fiber matrix of the invention has an
ability to absorb a liquid suspension readily and quickly, as well
as to release the biological specimen containing analytes of
interest consistently, efficiently, and precisely. In certain
embodiments, the polyolefin fiber matrix can absorb at least 0.05
ml, 0.1 ml, 0.2 ml, 0.3 ml, 0.4 ml, 0.5 ml, 0.6 ml, 0.7 ml, 0.8 ml,
or 0.9 ml, 1.0 ml, 1.5 ml, 2.0 ml, 2.5 ml, 3.0 ml, or greater,
sample of a liquid suspension of a biological specimen containing
an analyte of interest. The term "absorb" and "adsorb" are used
interchangeably, and means that the liquid suspension is
incorporated into or onto the polyolefin fiber matrix in such a way
as to be readily removed from the matrix leaving the analytes of
interest behind.
[0098] The volume of the polyolefin matrix may or may not expand
upon absorption of the liquid suspension, and may or may not
contract upon drying. However, a liquid saturated matrix can be
compressed to release entrained fluid containing analyte, due to
its porosity, by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%,
75%, 80%, 90%, or more of its saturated volume. Volumetric
compression is one convenient technique for release of the
reconstituted biological specimen, however, any other means, such
as centrifugation or vacuum pressure, can alternatively be employed
to release the biological specimen from the matrix.
[0099] Therefore, as used herein, the term "compress,"
"compressable," "compression," and other derivatives of the word
"compress" means that the volume of the saturated matrix is reduced
as compared to the original volume of the saturated matrix while a
force or a pressure is applied to the matrix. As used herein, the
term "a portion of the biological specimen" means at least some of
the biological specimen contained in the liquid suspension is
released from the matrix. In certain embodiments, the matrix is
compressed until the maximum volume of the reconstituted biological
specimen is released from the matrix.
[0100] In certain embodiments, the polyolefin fiber matrix is
three-dimensional in a shape such as cylinder, cube, sphere,
pyramid or cone. In certain embodiments, the matrix is in the shape
of a cylinder about 21 mm in length and 9 mm in diameter, with a
weight of about 0.103 grams. However, the matrix can be widened,
lengthened, or shortened to achieve any needed volume capacity.
Polyolefin fiber sizes can vary, but are generally about 1-100.mu.
or 20-25 microns.
[0101] In certain embodiments, for reconstitution and recovery of
the analytes the matrix is mounted or placed within a container or
syringe barrel into which is received a plunger, wherein the matrix
is compressed by applying force to the plunger against the matrix
to release reconstituted biological suspension through a port for
example. In yet other embodiments, the matrix can be removable from
the enclosed container and the plunger. As used herein, the term
"removable" means that the matrix can be detached or separated from
the container and the plunger.
[0102] As used herein, the term "liquid suspension" refers to any
liquid medium and mixture containing biological specimens. This
includes, for example, water, saline; cell suspensions of humans,
animals and plants; extracts or suspensions of bacteria, fungi,
plasmids, viruses; extracts or suspensions of parasites including
helminthes, protozoas, spirochetes; liquid extracts or homogenates
of human or animal body tissues, e.g., bone, liver, kidney, brain;
media from DNA or RNA synthesis; mixtures of chemically or
biochemically synthesized DNA or RNA, and any other sources in
which any biological specimen is or can be in a liquid medium.
[0103] As used herein, the term "biological specimen" refers to
samples, either in liquid or solid form, having dissolved,
suspended, mixed or otherwise contained therein, any analytes of
interest, for example, genetic material. As used herein, the term
"genetic material" refers to nucleic acids that include either or
both deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). The
term "biological specimen" also refers to whole blood, plasma,
serum lymph, synovial fluid, bone marrow, cerebrospinal cord fluid,
semen, saliva, urine, feces, sputum, vaginal lavage, skin
scrapings, hair root cells, or the like of humans or animals,
physiological and pathological body liquids, such as secretions,
excretions, exudates and transudates; any cells or cell components
of humans, animals, plants, bacteria, fungi, plasmids, viruses,
parasites, or the like that contain analytes of interest, and any
combination thereof.
[0104] As used herein, the term "analytes of interest" refers to
any micro- or macro-molecules in the biological specimen that are
interested to be detected or analyzed. These include, for example,
nucleic acids, polynucleotides, oligonucleotides, proteins,
polypeptides, oligopeptides, enzymes, amino acids, receptors,
carbohydrates, lipids, cells, any intra- or extra-cellular
molecules and fragments, virus, viral molecules and fragments, or
the like. In certain embodiments, the analytes of interest are
nucleic acids including either or both DNA or RNA. As used herein,
the term "nucleic acids" or "polynucleotide" refers to RNA or DNA
that is linear or branched, single or double stranded, a hybrid, or
a fragment thereof. The term also encompasses RNA/DNA hybrids. The
term also encompasses coding regions as well as upstream or
downstream noncoding regions. In addition, polynucleotides
containing less common bases, such as inosine, 5-methylcytosine,
6-methyladenine, hypoxanthine, and other are also encompassed.
Other modifications, such as modification to the phosphodiester
backbone, or the 2'-hydroxy in the ribose sugar group of the RNA
are also included. The nucleic acids/polynucleotides may be
produced by any means, including genomic preparations, cDNA
preparations, in vitro synthesis, RT-PCR, and in vitro or in vivo
transcription. In certain embodiment, the nucleic acids are either
or both viral DNA or RNA, for example, DNA or RNA from human
immunodeficiency virus (HIV), hepatitis B virus (HBV), hepatitis C
virus (HCV), or any other human or animal viral pathogen.
[0105] In certain embodiments, the compression device provided by
the present invention may optionally include a desiccant, either a
natural or synthetic desiccant, inside the container to maintain
the dried state of the matrix and integrity of the analytes of
interest on the matrix within the enclosed container. In certain
embodiments, the desiccant is in vaporous communication with the
matrix in the compression device having a dye indicator reactive
with moisture whereby the desiccant changes to a bright color when
exposed to humidity or moisture. In certain embodiments, the
desiccant is in vaporous communication with the matrix so that an
air permeable barrier is formed in-between the desiccant and the
matrix inside the container. The desiccant used in the device is
commonly known in the art, including but is not limited to
montmorillonite clay, lithium chloride, activated alumina, alkali
alumino-silicate, DQ11 Briquettes, silica gel, molecular sieve,
calcium sulfate, and calcium oxide. The desiccant can be provided
with a colorimetric indicator of water content. A desiccant may not
be needed inside the device with the hydrophobic polyolefin fiber
matrix of the invention.
[0106] The polyolefin fiber matrix of the invention may optionally
include a composition absorbed to the matrix wherein the
composition protects against degradation of the analytes of
interest contained in the biological specimens. As used herein, the
term "protects against degradation of the analytes of interest"
means that a matrix in the device of the invention maintains the
stored analytes of interest contained in the biological specimens
in a substantially nondegraded form, providing that the analytes of
interest are suitable for many different types of subsequent
analytical procedures. Protection against degradation may include
protection against substantial damaging of analytes of interest
caused by chemical or biological agents including action of
bacteria, free radicals, nucleases, ultraviolet radiation,
oxidizing agent, alkylating agents, or acidic agents (e.g.,
pollutants in the atmosphere). In certain embodiments, the
composition absorbed on the matrix of the invention may include one
or more of a weak base, a chelating agent, a protein denaturing
agent such as a detergent or surfactant, a nuclease inhibitor, and
a free radical trap. In the case where the stored analyte of
interest is RNA, particularly unstable RNA, the composition may
include RNase inhibitors and inactivators, genetic probes,
complementary DNA or RNA (or functionally equivalent compounds),
proteins and organic moieties that stabilise RNA or prevent its
degradation.
[0107] Another composition which protects against degradation which
may be optionally used is an oxygen scavenger element. As used
herein, the term "oxygen scavenging element" refers to is a
substance that consumes, depletes or reduces the amount of oxygen
from a given environment without negatively affecting the samples
of interests. Suitable oxygen scavenging elements are well-known to
those skilled in the art. Non-limiting examples of oxygen
scavenging elements include but are not limited to compositions
comprising metal particulates reactive with oxygen such as
transition metals selected from the first, second or third
transition series of the periodic table of the elements, and
include manganese II or III, iron II or III, cobalt II or III,
nickel II or III, copper I or II, rhodium II, III or IV, and
ruthenium. The transition metal is preferably iron, nickel or
copper. An example of an iron oxygen scavenging element is D500
from Multisorb. Other commercially available oxygen scavengers may
also be purchased from companies such as Mitsubishi, Dow, or the
like. Other examples of oxygen scavenging element may be enzymes
which consumes, depletes or reduces the amount of oxygen from the
given environment without negatively affecting the samples of
interests.
[0108] In other embodiments, the compression device may optionally
comprise a modified atmosphere such as nitrogen or argon through a
well-known gas purging process prior to sealing, shipping, or
storing. The term "modified atmosphere" refers to any replacing or
altering normal atmospheric gas compositions with at least one
inert gas or gas which does not degrade the sample of
interests.
[0109] As used herein, a "weak base" suitable for the composition
of the invention may be a Lewis base which has a pH of about 6 to
10, preferably about pH 8 to 9.5. The weak base suitable for the
composition of the invention may, in conjunction with other
components of the composition, provide a composition pH of 6 to 10,
preferably, about pH 8.0 to 9.5. Suitable weak bases according to
the invention include organic and inorganic bases. Suitable
inorganic weak bases include, for example, an alkali metal
carbonate, bicarbonate, phosphate or borate (e.g., sodium, lithium,
or potassium carbonate). Suitable organic weak bases include, for
example, tris-hydroxymethyl amino methane (Tris), ethanolamine,
triethanolamine and glycine and alkaline salts of organic acids
(e.g., trisodium citrate). A preferred organic weak base is a weak
monovalent organic base, for example, Tris. The weak base may be
either a free base or a salt, for example, a carbonate salt. It is
believed that the weak base may provide a variety of functions,
such as protecting the analytes of interest from degradation,
providing a buffer system, ensuring proper action of the chelating
agent in binding metal ions, and preventing the action of acid
nucleases which may not be completely dependent on divalent metal
ions for functioning.
[0110] As used herein, a "chelating agent" is any compound capable
of complexing multivalent ions including Group II and Group III
multivalent metal ions and transition metal ions (e.g., Cu, Fe, Zn,
Mn, etc). In certain embodiments, the chelating agent is ethylene
diamine tetraacetic acid (EDTA), citrate or oxalate. It is believed
that one function of the chelating agent is to bind multivalent
ions which if present with the stored biological specimen may cause
damage to the analytes of interest, especially to nucleic acids.
Ions which may be chelated by the chelating agent include
multivalent active metal ions, for example, magnesium and calcium,
and transition metal ions, for example, iron. Both calcium and
magnesium are known to promote nucleic acid degradation by acting
as co-factors for enzymes which may destroy nucleic acids (e.g.,
most known nucleases). In addition, transition metal ions, such as
iron, may readily undergo oxidation and reduction and damage
nucleic acids by the production of free radicals or by direct
oxidation.
[0111] The composition can further include a protein denaturing
agent if the analytes of interest are nucleic acids. As used
herein, a "protein denaturing agent" functions to denature
non-nucleic acids compounds, for example, nucleases. If the protein
denaturing agent is a detergent or a surfactant, the surfactant may
also act as a wetting agent to facilitate the uptake of a sample by
the dry solid matrix. The terms "surfactant" and "detergent" are
synonymous and may be used interchangeably throughout the
specification. Any agent that denatures proteins without
substantially affecting the nucleic acids of interest may be
suitable for the invention. In certain embodiments, protein
denaturing agents include detergents. As used herein "detergents"
include ionic detergents, preferably anionic detergents. An anionic
detergent suitable for the invention may have a hydrocarbon moiety,
such as an aliphatic or aromatic moiety, and one or more anionic
groups. Particularly, suitable anionic detergents include sodium
dodecyl sulphate (SDS) and sodium lauryl sarcosinate (SLS). The
ionic detergent causes inactivation of a microorganism which has
protein or lipid in its outer membranes or capsids, for example,
fungi, bacteria or viruses. This includes microorganisms which may
be pathogenic to humans or which may cause degradation of nucleic
acids. It is believed that inactivation of a microorganism by a
detergent is a result of destruction of the secondary structure of
the organisms external proteins, internal proteins, protein
containing membranes, or any other protein necessary for viability.
However, the detergent may not inactivate some forms of organisms,
for example, highly resistant bacterial spores and extremely stable
enteric virions.
[0112] The composition may optionally include a free radical trap.
As used herein, a "free radical trap" is a compound which is
sufficiently reactive to be preferred, over a DNA molecule or a
component thereof, as a reactant with a free radical, and which is
sufficiently stable not to generate damaging free radicals itself.
Examples of a suitable free radical trap include: uric acid or a
urate salt, mannitol, benzoate (Na, K, Li or tris salt), 1-3
dimethyl uric acid, guanidine, guanine, thymine, adenine, cytosine,
in N-acetyl-histidine, histidine, deferoxamine, dimethyl sulfoxide,
5'5' dimethyl pyrroline-N-oxide, thiocyanate salt and thiourea.
Suitable free radical traps include mannitol, thiocyanate salts,
uric acid or a urate salt. It is believed that the longer the
period of time for which the nucleic acid is to be stored the more
likely that a free radical trap may be advantageously included in
the composition absorbed to the solid matrix. Even if the nucleic
acid is only to be stored for a matter of minutes, a free radical
trap may still be incorporated into the composition. It is believed
that one function of the free radical trap may be to trap nucleic
acid damaging free radicals. For example, when the free radical
trap used is uric acid or urate salt it may be converted to
allantoin which may also act as a free radical trap that accepts
free radicals that would otherwise damage nucleotide bases, for
example, guanine. In certain embodiments, the free radical trap
reacts with free radicals regardless of source (including free
radicals present in the air). Free radicals may be generated
through oxidation or reduction of iron in biological specimen, such
as blood. Typically, free radicals are believed to be generated by
spontaneous oxidation of the groups which are present, for example,
in denatured serum protein of blood. Free radicals may also be
generated by radiation such as UV light, x-rays and high-energy
particles. In addition, free radical traps which are also a weak
acid, e.g. uric acid, may also function as a component of the
buffering system provided by the weak base discussed above. Also,
the free radical trap may enhance removal of a stored sample of
nucleic acids if in situ processing is not desired.
[0113] Referring to FIGS. 1A & 1B, an exemplary compression
device of the invention for preserving liquid suspension of
biological specimen containing analytes of interest is shown. The
container 20 is cylindrical and has side walls 22, a bottom 24 and
an openable lid 26, which sealingly engages the container 20
opening. The lid 26 has an extension 28 that holds a removable
matrix 30 inside the container 20. The polyolefin fiber matrix 30
is a cylinder capable of absorbing 1 ml of a liquid suspension of a
biological specimen and compress by at least 50% of the volume of
the saturated matrix to release a portion of the biological
specimen. A desiccant 40 may be optionally placed inside the
container 20, separated with the matrix 30 by an optional air
permeable barrier 42, for in vaporous communication with the matrix
30 to control humidity or moisture therein.
[0114] The invention further provides a method for preserving and
recovering a biological specimen comprising: (a) providing a dried
biological specimen in a device comprising a container defining an
interior space having side walls, a bottom and an openable and
sealable lid with an absorbent three-dimensional polyolefin matrix
mounted inside the container, wherein the polyolefin matrix
comprises a plurality of interstices with a hydrophobic polyolefin
surface and has contained therein the dried biological specimen
obtained from an evaporated volume of at least 0.1 ml of a liquid
suspension comprising a solvent and the biological specimen
absorbed and dried on the matrix; (b) reconstituting the biological
specimen on the polyolefin matrix with a controlled volume of a
reconstitution media; and (c) removing the biological specimen and
reconstitution media from the polyolefin matrix by compressing the
matrix. Any suitable and/or commonly available drying methods, such
as vacuum dry, low heat dry, low pressure dry, and fan dry, can be
used in the inventive method.
[0115] In certain embodiments, the polyolefin matrix comprises a
plurality of fibers having a substantially hydrophobic surface. In
certain embodiments, the fibers within the polyolefin matrix have a
polyethylene surface. In other embodiments, the fibers within the
polyolefin matrix comprise polypropylene coated with polyethylene.
In certain embodiments, the polypropylene and polyethylene are
present in approximately equal amounts in each fiber strand.
[0116] Referring to FIG. 1B, the lid 26 of the container 20 has a
lid extension 28 holding a polyolefin fiber matrix 30 which may be
permanently mounted in a cup that is attached to a plunger
contained within the second enclosed container. A liquid suspension
of any biological specimen containing analytes of interest is added
on the top of the polyolefin fiber matrix 30 and is allowed to
fully absorb into the matrix 30 (FIG. 3). The lid 26 with the
matrix 30 having bound biological specimen thereon is allowed to
air-dry, and then reassembled with the container 20 for
preservation at ambient temperature.
[0117] The method of the invention further optionally includes an
intermediate step of applying a stabilizing composition to the
polyolefin fiber matrix to protect the analytes of interest against
degradation. Depending upon the analytes of interest, the
stabilizing composition, as discussed above, may include but is not
limit to one or more of a weak base, a chelating agent, a protein
denaturing agent such as a detergent or surfactant, a nuclease
inhibitor, and a free radical trap. Particularly for protection of
unstable RNA, the stabilizing composition may include RNase
inhibitors and inactivators, genetic probes, complementary DNA or
RNA (or functionally equivalent compounds), proteins and organic
moieties that stabilise RNA or prevent its degradation.
[0118] The invention further provides a method for recovering from
the polyolefin fiber matrix in the compression device the
biological specimen containing analytes of interest. In certain
embodiments, the method includes the following steps: a) applying
reconstitution medium to the matrix to rehydrate the bound
biological specimen containing analytes of interest, and b)
compressing the matrix to release a portion of the biological
specimen. According to the present invention, the reconstitution
medium is molecular-grade water. In other embodiments, the
reconstitution medium includes the components of 1.times. phosphate
buffered saline (PBS) or nuclease-free water optionally with the
addition of sodium azide or other antimicrobial agent. The
reconstitution medium may also include any number or combinations
of available biological preservatives or blood anticoagulants
including but not limited to ethylenediaminetetraacetic acid
(EDTA), sodium citrate, and heparin. PBS or nuclease-free water
serves as the sterile and neutral medium for the rehydration,
resuspension, and recovery of the analyte(s) of interest from the
matrix. When included, antimicrobial agents such as sodium azide
prevent microbial growth and subsequent contamination with RNases.
When included, biological preservatives such as EDTA, sodium
citrate, and heparin serve as anticoagulants and or chelating
agents.
[0119] In the embodiments shown in FIGS. 4-7, the biological sample
is prepared for analysis. FIG. 4 is a perspective view of preparing
to transfer the polyolefin fiber matrix 30 of the device to an
empty syringe barrel 52. FIG. 5 is a perspective view of completed
delivery of the polyolefin fiber matrix 30 into the syringe barrel
52.
[0120] FIG. 6 illustrates rehydration of the polyolefin fiber
matrix 30 by a pipette tip 53 gently placed on the top of the
matrix 30 and slowly dispensing reconstitution buffer. FIG. 7A
illustrates insertion of the plunger 54 into the syringe barrel 54.
FIG. 7B illustrates application of pressure to the syringe plunger
42. FIG. 7C illustrates compression of the matrix 30. FIG. 7D
illustrates completion of sample recovery from the matrix 30.
[0121] In certain embodiments, the analytes of interest are nucleic
acids including either or both DNA or RNA molecules. In certain
embodiments, the liquid suspension of biological specimen contains
at least about 5 attograms or 1 .mu.g isolated DNA or RNA
molecules. As used herein, the term "isolated," "isolation," and
other derivatives of the word "isolate" means that the DNA or RNA
molecules are substantially free from some of the other cellular
material with which it is naturally associated, or culture medium
when produced by recombinant techniques, or chemical precursors or
other chemicals when chemically synthesized.
[0122] The invention further provides that the analytes of interest
contained in the biological specimen recovered from the polyolefin
fiber matrix of the device into the reconstitution medium, such as
molecular-grade water, are subject to subsequent analysis. As used
herein, the term "subsequent analysis" includes any analysis which
may be performed on recovered biological specimens stored in
reconstitution medium. Alternatively, the analytes of interest
contained in the biological specimen may be isolated, purified or
extracted prior to analysis using methods known in the art. The
analytes of interest may be subjected to chemical, biochemical or
biological analysis. In one of the preferred embodiments, the
analytes of interest are nucleic acids including either or both DNA
or RNA molecules that can be detected or analyzed with or without
prior extraction, purification or isolation. DNA or RNA extraction,
purification or isolation, if necessary, is performed based on
methods known in the art. Examples of subsequent analysis include
polymerase chain reaction (PCR), ligase chain reaction (LCR),
reverse transcriptase initiated PCR, DNA or RNA hybridization
techniques including restriction fragment length polymorphism
(RFLP), viral DNA or RNA detection and quantification, viral load
tests, DNA or RNA genotyping, etc. "Subsequent analysis" also
includes other techniques using genetic probes, genomic sequencing,
enzymatic assays, affinity labeling, methods of detection using
labels or antibodies and other similar methods.
[0123] The invention also provides a kit for preserving a liquid
suspension of biological specimen containing analytes of interest.
The kit of the invention provides a compression device disclosed
herein including one or more containers, one or more polyolefin
fiber matrixes, and optionally desiccant, and instructions for the
use thereof to preserve biological specimens. The kit may
optionally include a stabilising solution. Kits of the invention
can further include a reconstitution medium, a compression device
and further protocols for rehydration and recovery of the
biological specimen. The container of the kit may be any container
suitable for use during application of a liquid suspension of
biological specimen containing analytes of interest to the matrix
or during application and one or more phases of subsequent
processing of a sample of the biological specimen. Therefore, in
certain embodiments, a liquid suspension of biological specimen may
be applied, stored, transported and further processed all in the
same kit. Alternatively, a liquid suspension may be applied to the
matrix where the matrix is removed from the kit container for
processing in a different container.
[0124] The kit may also include one or more of any of the
polyolefin fiber matrix disclosed herein. This includes one or more
polyolefin fiber matrix with or without compositions for protection
of analytes of interest contained in the biological specimen. One
aspect of the kit of the invention is that the reconstituted
biological specimen containing analytes of interest is released by
compressing the matrix. This procedure avoids vortexing and
centrifuging the sample, providing decreased chance of sample
damage, human labor costs and matrix contamination of the sample. A
compression device of the kit of the present invention may be any
device that is used to provide a force or pressure on the matrix to
compress it. In certain embodiments, the compression device
comprising a plunger permanently attached to the polyolefin fiber
matrix, wherein the matrix is compressed by applying force to the
plunger against the matrix in the same kit container(s) where
biological specimens are prepared and stored in. Alternatively, the
compression device comprises a syringe separate from the polyolefin
fiber matrix, wherein the matrix is removed from the container and
placed in the syringe barrel and the force or pressure is applied
to the plunger of the syringe to compress the matrix to release the
reconstituted biological specimen.
[0125] Throughout this application, various publications are
referenced. The disclosures of all of these publications and those
references cited within those publications in their entireties are
hereby incorporated by reference into this application in order to
more fully describe the state of the art to which this invention
pertains.
[0126] It should also be understood that the foregoing relates to
certain embodiments of the present invention and that numerous
changes may be made therein without departing from the scope of the
invention. The invention is further illustrated by the following
examples, which are not to be construed in any way as imposing
limitations upon the scope thereof. On the contrary, it is to be
clearly understood that resort may be had to various other
embodiments, modifications, and equivalents thereof, which, after
reading the description herein, may suggest themselves to those
skilled in the art without departing from the spirit of the present
invention and/or the scope of the appended claims.
EXAMPLES
Example 1
One (1.0) ml Sample Preparation and Device Recovery Kit
Kit Components:
[0127] This example provides a kit for the preparation,
transportation, and recovery of thirty-six (36) dry biological
specimens from bodily fluids or tissue. Materials and reagents for
the preparation and recovery of thirty-six (36) one (1.0) ml
samples for dried ambient transportation include the following:
TABLE-US-00001 Component Quantity Device Kit containers (tubes) 36
each Reconstitution Buffer 3 .times. 13 ml Disposable 3 ml Syringes
36 each 15 ml Conical Centrifuge Tubes 36 each
Storage and Handling:
[0128] Upon receipt, all kit components are stored dry at room
temperature (15-25.degree. C.). Only use device container tubes
when the indicating desiccant is blue in color. The device kit
container tubes should not be if the indicating desiccant appears
white or pink in color. Materials, such as 1000 .mu.l pipette, 1000
.mu.l sterile DNase-free, RNase-free pipette tips with aerosol
barrier, rack for holding 15 ml conical tubes, safety glasses,
laboratory coat, powder-free disposable gloves and biohazard waste
container, are also required but are not provided by the kit Safety
Precautions: Disposable powder-free gloves are used to handle all
materials as though capable of transmitting infectious agents.
Utilise good laboratory practices and universal precautions
relating to the prevention of transmission of blood borne pathogens
(Centers for Disease Control. Update: Universal precautions for
prevention of transmission of human immunodeficiency virus,
hepatitis B virus and other blood borne pathogens in healthcare
settings. MMWR, 1988; 37: 377-82, 387-8; National Committee for
Clinical Laboratory Standards. Protection of laboratory workers
from infectious disease transmitted by blood, body fluids, and
tissue; approved guideline. NCCLS Document M29-A Villanova (PA):
NCCLS; 1997 December 90p; Federal Occupational Safety and Health
Administration. Bloodborne Pathogens Standard, 29 CFR 1910, 1030).
Any spills suspected of potentially containing infectious agents
were immediately cleaned up with 0.5% w/v sodium hypochlorite (10%
v/v bleach). Dispose of all specimens and materials coming into
contact with specimens as though they contain infectious agents. In
the event that materials known or suspected of containing
infectious agents are ingested or come in contact with open
lacerations, lesions, or mucous membranes (eyes, nasal passages,
etc.), consult a physician immediately.
Example 2
Sample Preparation Using the Device Kit
[0129] The sample preparation steps were performed within a
biological safety cabinet using sterile technique and universal
precautions relating to the handling of potentially infectious
materials. Before beginning the sample preparation process, the
protocol of using the device kit that is illustrated in FIGS. 1A
& 1B should be familiarized.
[0130] Before loading a sample liquid suspension of biological
specimen containing analytes of interest, the cap from the device
container was unscrewed, inverted and placed on a clean working
surface with the absorbent matrix facing upwards (FIGS. 1A &
1B). About up to 1 ml of sample fluid was slowly added to the top
of the matrix plug and allowed it to fully absorb into the matrix.
The device kit matrix loaded with the sample fluid was allowed to
air-dry. In general, air-drying within a biological safety cabinet
takes approximately 4.5 to 5 hours. Once the sample is completely
dry, the cap holding the dried specimen-containing matrix was
carefully reattached back to the device kit container tube. The
specimen is now ready for shipment or storage at ambient
temperature.
Example 3
Sample Recovery Using the Device Kit
[0131] The sample recovery steps were also performed within a
biological safety cabinet using sterile technique and universal
precautions relating to the handling of potentially infectious
materials. Basically, a sterile 3 or 5 ml disposable LUER-LOK
syringe (provided by the kit) was inserted into a 15 ml collection
tube (also provided by the kit). The plunger was removed from the
syringe barrel. The absorbent matrix containing the dried specimen
was transferred into the syringe barrel by pressing the matrix
against the sterile inside of the syringe barrel's mouth with just
enough pressure to break it free from the attached cap and allow it
to fall freely to the bottom of the syringe (FIGS. 4 & 5). The
syringe barrel with detached matrix plug was placed into a 15 ml
conical collection tube, which is further placed into a rack. About
1 ml of Reconstitution Buffer (supplied by the kit) was applied
slowly and directly to the top of the matrix plug to gently
re-hydrate the dried specimen absorbed inside the matrix (FIG. 6).
It is necessary to inspect the absorption rate and adjust the
application speed as needed while adding the reconstitution buffer,
and try not to allow buffer to collect at the bottom of the syringe
without first being absorbed into the matrix because failing to
fully absorb the reconstitution buffer may result in lower recovery
yields. The re-hydrating specimen was allowed to incubate for at
least 10 minutes at room temperature prior to adding an additional
175 .mu.l of Reconstitution Buffer to the top of the matrix
plug.
[0132] The syringe plunger was re-inserted into the syringe barrel
and depressed with firm even pressure until the plunger has
completely compressed the matrix plug and a maximum volume of
approximately 1 ml is collected inside the 15 ml collection tube
(FIG. 7A, 7B, 7C, & 7D). The syringe barrel, the plunger and
the compressed matrix plug were then removed from the 15 ml
collection tube and discarded into an appropriate waste receptacle.
The 15 ml collection tube containing the newly recovered specimen
was sealed with the provided screw cap. The reconstituted sample is
ready for storage, testing, or further subsequent analysis.
Example 4
ViveST Device Matrix Comparison Study
1. Purpose
[0133] The purpose of this study was to compare performance of the
ViveST devices with the cellulose matrix to the ViveST devices of
the invention with the synthetic hydrophobic polyolefin fiber
matrix using the Abbott REALTIME HBV assay. HBV infectious samples
(3 levels, 5 replicates each) were loaded and stored for 7 days at
ambient conditions on both matrixes. Specimens recovered from the
matrixes were run simultaneously with frozen samples.
2. Methodology
[0134] All testing on the Abbott REALTIME HBV assay was performed
according to the FDA approved protocol (0.5 mL) with no
modifications. 1 mL HBV infectious plasma was loaded onto each
matrix, stored for 7 days at ambient temperature and recovered in 1
mL molecular grade water.
[0135] HBV viral load results of frozen samples (3 levels, 5
replicates each) were compared to specimen recovered from the
ViveST devices with cellulose matrix and the ViveST devices of the
invention having the hydrophobic polyolefin fiber matrix.
3. Equipment and Reagents
[0136] The following commercially available products/equipments
were utilised in the course of this study: ViveST sample storage
and transportation devices of the invention (Catalogue No. VST-1E,
ViveBio LLC, Alpharetta, Ga.), and the ViveST devices with the
cellulose matrix (ViveBio, LLC, Alpharetta, Ga.); BD 3 mL
syringe-LUER-LOK Tip: Ref 3096567 (Becton Dickenson; Franklin
Lakes, N.J.); General lab consumables and equipment (centrifuge
tubes, sterile aerosol resistant pipette tips, pipettes, vortex,
centrifuge, etc.; HYCLONE HYPURE molecular biology grade water
(Catalogue No.: SH30538.02, HyClone Laboratory Inc., Logan, Utah);
Human plasma (Tennessee blood services; Memphis, Term.); Abbott
sample preparation system (4.times.24 Preps), List number:
06K12-024 (Abbott Molecular Inc; Des Plaines, Ill.); Abbott
REALTIME HBV AMP kit (Catalogue number: 02N40-90, Abbott Molecular
Inc; Des Plaines, Ill.); Abbott REALTIME HBV Control kit (Catalogue
number: 02N40-80, Abbott Molecular Inc; Des Plaines, Ill.); Abbott
REALTIME HBV Calibration kit (Catalogue number: 02N40-70, Abbott
Molecular Inc; Des Plaines, Ill.); Abbott m2000sp System including
m2000rt (Abbott Molecular Inc; Des Plaines, Ill.), and associated
materials.
4. Experimental Design
[0137] A high titer HBV infectious plasma sample was diluted in
normal human plasma to yield 3 concentrations (.about.5 LOG,
.about.4 LOG and .about.3 LOG). 5 replicates of each concentration
(n=15 total) were loaded onto each of the ViveST devices with the
cellulose matrix and the ViveST devices of the invention with the
polyolefin fiber matrix. Identical aliquots (3 levels, 5
replicates) were stored frozen (-80.degree. C.). All matrixes were
dried overnight in a laminar flow hood, capped the following day
and stored at ambient conditions for 7 days. Specimens were
recovered from both sets of matrixes and analyzed concurrently with
the frozen specimens in a single assay run as outlined in the
Abbott REALTIME HBV assay package insert and in accordance with the
bioMONTR Research Method (RM-008.00, Quantitation of HBV RNA using
the Abbott REALTIME HBV assay).
5. Results--A Summary of the Abbott REALTIME HBV Viral Load Results
is Provided in Table 1 Below.
TABLE-US-00002 [0138] TABLE 1 Summary of ViveST Matrix Comparison
Study Data Achieved HBV Concentration (LOG IU/mL) Target HBV Target
HBV 2.sup.nd 1.sup.st Concentration Concentration Generation
Generation Level (LOG IU/mL) (IU/mL) Replicate Frozen Matrix Matrix
1 5.00 100,000 a 4.94 4.93 4.69 b 4.90 4.93 4.69 c 5.02 4.91 4.55 d
5.00 4.89 4.73 e 4.99 4.90 4.54 Average 4.97 4.91 4.64 Std Dev 0.05
0.02 0.09 95% CI 0.04 0.02 0.08 2 4.00 10,000 a 3.96 3.86 3.76 b
3.93 3.92 3.73 c 3.93 3.86 3.59 d 3.88 3.86 3.80 e 3.84 3.87 3.64
Average 3.91 3.87 3.70 Std Dev 0.05 0.03 0.09 95% CI 0.04 0.02 0.08
3 3.00 1,000 a 2.81 2.90 2.78 b 2.94 2.93 2.74 c 2.85 2.90 2.80 d
2.97 2.87 2.67 e 2.94 2.88 2.61 Average 2.90 2.90 2.72 Std Dev 0.07
0.02 0.08 95% CI 0.06 0.02 0.07
[0139] Results of this example demonstrated an average reduction
across all concentrations of: a) 0.03 LOG IU/mL between the frozen
plasma and the plasma samples stored on the ViveST devices of the
invention with the polyolefin fiber matrix; and b) 0.24 LOG IU/mL
between the frozen plasma and the plasma samples stored on the
ViveST devices with the cellulose matrix. The Standard Deviations
(LOG IU/mL) across all concentrations were: a) <0.07 for the
frozen plasma; b) <0.03 for the plasma samples stored on the
ViveST devices of the invention with the polyolefin fiber matrix;
and c) <0.09 for the plasma samples stored on the ViveST devices
with the cellulose matrix.
[0140] As shown in FIG. 8, the linear regression analysis yielded:
a) R.sup.2=0.999998 for the frozen plasma as compared to the plasma
samples stored on the ViveST devices of the invention with the
polyolefin fiber matrix; and b) R.sup.2=0.9991 for the frozen
plasma as compared to the plasma samples stored on the ViveST
devices with the cellulose matrix.
6. Final Conclusion
[0141] Therefore, this example provides that HBV infectious plasma
samples stored on the ViveST devices of the invention with the
polyolefin fiber matrix were recovered and yielded results similar
to the frozen plasma. There was minimal loss (0.03 LOG IU/mL) when
compared to frozen plasmas and very high reproducibility across all
concentrations (Std Dev<0.03). In contrast, HBV infectious
plasma samples stored on the ViveST devices with the cellulose
matrix exhibited greater loss as compared to the frozen plasma
(0.24 LOG IU/mL) and a higher variability across all concentrations
(Std Dev<0.09).
[0142] Therefore, the ViveST devices of the invention with the
polyolefin fiber matrix provide better and superior sample recovery
and minimized sample loss, as well as providing reproducibility
across all concentration, as compared to the devices with the
cellulose matrix, suggesting that the polyolefin fiber matrix
retains analytes and suspended particles inside the matrix better
than the cellulose matrix, and allows the solvents to evaporate
more consistently and efficiently.
Example 5
Extracted RNA Versus Intact Virus
1. Experimental Design
[0143] The purpose of this experiment was to evaluate polyolefin
matrix ViveST devices' binding and releasing properties of nucleic
acid as compared to intact virus. 1 mL aliquots of HCV infectious
plasma samples (N=20), stored at -80.degree. C., were used for the
study and were designated as follows:
[0144] A=RNA was extracted using the EasyMAG system and loaded on
the ViveST devices with the cellulose fiber matrix, and then
recovered with water and analyzed with the Abbott REALTIME HCV
assay;
[0145] B=RNA was extracted using the EasyMAG system and loaded on
the ViveST devices of the invention with the polyolefin fiber
matrix, and then recovered with water and analyzed with the Abbott
REALTIME HCV assay;
[0146] C=Sample loaded on the ViveST devices with the cellulose
fiber matrix, and recovered with water and analyzed with the Abbott
REALTIME HCV assay; and
[0147] D=Sample loaded on the ViveST devices of the invention with
the polyolefin fiber matrix, and recovered with water and analyzed
with the REALTIME Abbott HCV assay.
2.1 Procedure--Nucleic acid isolation using EasyMAG followed by
processed through either the ViveST devices with the cellulose
fiber matrix or the polyolefin fiber matrix.
[0148] a). Removed 1 mL plasma aliquots designated as "1A, 2A . . .
20A" and "1B, 2B . . . 20B" from -80.degree. C., thawed at room
temperature;
[0149] b). Vortexed each sample to ensure adequate mixing;
[0150] c). Following the EasyMAG standard nucleic acid extraction
protocol, processed the 40 samples +2 negative controls, after
extraction, all samples were diluted 1 mL volume using EasyMAG
elution buffer;
[0151] d). Obtained 42 ViveST devices and labeled the cap of each
with the sample designations (i.e., 1A-20A, 1B-20B, neg
control--old matrix, neg control--new matrix);
[0152] e). Loaded 1 ViveST device for each sample;
[0153] f). Dried the loaded matrix in the ViveST devices in a
laminar flow hood for at least 12 hours but not more than 36
hours;
[0154] g). Recovered samples from the dried ViveST devices using
water; and
[0155] h). Froze the recovered samples at -80.degree. C. prior to
proceeding to the Abbott REALTIME HCV assay.
2.2 Procedure--Sample processed through the ViveST devices with
cellulose fiber matrix or the polyolefin fiber matrix
[0156] a) Removed 1 mL plasma aliquots designated as "1C, 2C . . .
20C" and "1D, 2D . . . 20D" from -80.degree. C., thawed at room
temperature;
[0157] b) Vortexed each sample to ensure adequate mixing;
[0158] c) Obtained 42 ViveST devices and labeled the cap of each
with the sample designations (i.e., 1C-20C, 1D-20D, neg
control--cellulose matrix, neg control--polyolefin fiber
matrix);
[0159] d) Loaded 1 ViveST device for each sample;
[0160] e) Dried the loaded matrixes of the ViveST devices in a
laminar flow hood for at least 12 hours but not more than 36
hours;
[0161] f) Recovered samples from the dried ViveST devices using
water; and
[0162] g) Froze the recovered samples at -80.degree. C. prior to
proceeding to the Abbott's REALTIME HCV assay.
2.3. Procedure--Abbott REALTIME HCV Assay: Processed the samples
following the Abbott REALTIME HCV package insert.
3. Results: The Results are Provided in the Following Table 2.
TABLE-US-00003 [0163] TABLE 2 Aliquot A Aliquot B Aliquot C Aliquot
D Extracted RNA loaded Extracted RNA loaded Plasma sample loaded
Plasma sample loaded on ViveST (old matrix), on ViveST (new
matrix), on ViveST (old matrix), on ViveST (old matrix), recovered
and analyzed recovered and analyzed recovered and analyzed
recovered and analyzed with Abbott RealTime with Abbott RealTime
Recovery of with Abbott RealTime with Abbott RealTime Recovery of
HCV Assay HCV assay Extracted RNA HCV Assay HCV Assay Whole Virus
HCV Results HCV Results % Difference HCV Results HCV Results %
Difference Sample LOG (IU/mL) LOG (IU/mL) (new vs old) LOG (IU/mL)
LOG (IU/mL) (new vs old) Neg TND TND TND TND Control 1 3.22 2.85**
Not Calculated 3.72 3.69 -0.81 3 3.71 3.84 3.39 3.86 3.63 -6.34 5
3.56 3.69 3.52 3.45 3.72 7.26 7 4.02 4.32 6.94 4.22 3.70 -14.05 8
4.22 4.48 5.80 4.30 4.15 -3.61 9 3.42 4.14 17.39 4.17 3.91 -6.65 10
2.01 2.40 16.25 2.73 2.36 -15.68 11 5.24 5.31 1.32 5.23 4.96 -5.44
12 4.24 4.55 6.81 4.58 4.17 -9.83 13 Error 4.09 Not Calculated 3.64
3.67 0.82 14 4.52 4.64 2.59 4.58 4.46 -2.69 16 4.54 4.47 -1.57 4.46
4.45 -0.22 17 3.85 4.39 12.30 4.38 4.49 2.45 19 4.24 4.53 6.40 4.33
4.12 -5.10 20 4.44 4.50 1.33 4.46 4.13 -7.99 21 4.65 4.74 1.90 4.61
4.60 -0.22 22 4.49 4.64 3.23 4.58 4.57 -0.22 23 4.20 4.58 8.30 4.61
4.54 -1.54 24 4.29 4.46 3.61 4.29 4.11 -4.38 25 3.03 3.50 13.43
3.37 3.12 -8.01 Average 3.99 4.28 6.62 4.18 4.03 -3.75 **Sample
Error on first m2000 run. The extracted RNA was rerun on the m2000.
Data not included in calculations.
4. Conclusions
[0164] On average, for Extracted RNA, the polyolefin fiber matrix
yielded higher recovery than the cellulose matrix (0.29 log IU/mL
higher). Near the clinically significant cut-off to support that
polyolefin matrix performs better for extracted RNA than the
cellulose matrix.
[0165] On average, the polyolefin fiber matrix yielded higher
recovery for extracted RNA as compared to the fresh plasma (0.25
log IU/mL higher). Near the clinically significant cut-off to
support that polyolefin matrix performs better for extracted RNA
than the fresh plasma.
[0166] Based on these data, the polyolefin fiber matrix is superior
as compared to the cellulose matrix for absorption, preservation,
stabilisation, and subsequent recovery of nucleic acid. These
surprising results are perhaps due to the properties of the
embedded hydrophobic pockets within the polyolefin matrix. These
pockets may provide a reservoir and `safe haven` for the nucleic
acid to reside while excluding the water from the nucleic acid
providing a stable environment for the nucleic acid during
storage.
Example 6
HCV Evaluation for Samples Processed Through the ViveST Devices of
the Invention
1. Experimental Design
[0167] 1 ml aliquots of HCV infectious plasma samples (N=19),
stored at -80.degree. C., was used for each part of the analysis
and was designated as follows:
[0168] A=sample analyzed with the Abbott REALTIME HCV assay
[0169] B=sample processed through the ViveST devices of the
invention with the polyolefin fiber matrix (elute with water) and
analyzed with the Abbott REALTIME HCV assay
[0170] C=sample analyzed with the Abbott HCV GT assay
[0171] D=sample processed through the ViveST devices of the
invention with the polyolefin fiber matrix (elute with water) and
analyzed with the Abbott HCV GT assay.
[0172] Additional aliquots of each plasma sample were maintained at
-80.degree. C. for additional testing.
2.1 Procedure--Sample processed through the ViveST devices of the
Invention
[0173] a) Removed plasma aliquots designated as "1B, 2B . . . 19B"
and "1D, 2D . . . 19D" from -80.degree. C., thawed at room
temperature;
[0174] b) Vortexed each sample to ensure adequate mixing;
[0175] c) Obtained 38 ViveST devices of the invention with the
polyolefin fiber matrix and labeled the cap of each with the sample
designations (i.e., 1B-19B, 1D-19D), obtained 2 additional ViveST
devices and labeled each as negative controls;
[0176] d) Loaded 1 ViveST device of the invention with the
polyolefin fiber matrix for each sample, loaded 1 mL normal (HCV
negative) human plasma on the ViveST devices labeled as negative
controls;
[0177] e) Dried the loaded matrix of the ViveST device in a laminar
flow hood for at least 12 hours but not more than 36 hours;
[0178] f) Recovered samples from the dried ViveST devices using
water; and
[0179] g) Froze recovered samples at -80.degree. C.
2.2 Procedure--Abbott m2000 REALTIME HCV assay
[0180] a) Removed plasma aliquots designated as "1A, 2A . . . 19A"
and the recovered aliquots from the ViveST devices of the invention
with the polyolefin fiber matrix as "1B, 2B . . . 19B and Neg
Control" from -80.degree. C., thawed at room temperature; and
[0181] b) Processed the samples following the Abbott REALTIME HCV
package insert.
2.3. Procedure--Abbott m2000 HCV GT Assay
[0182] a) Removed plasma aliquots designated as "1C, 2C . . . 19C"
and the recovered aliquots from the ViveST device of the invention
with the polyolefin fiber matrix as "1D, 2D . . . 19D and Neg
Control" from -80.degree. C., thawed at room temperature; and
[0183] b) Processed the samples following the Abbott HCV GT package
insert.
3. Results--A Summary of the Results is Provided in Tables 3 and 4
Below.
TABLE-US-00004 [0184] TABLE 3 Summary of the Abbot REALTIME HCV and
HCV GT Assay Results Aliquot B Aliquot D Process plasma Process
plasma samples Aliquot A samples through Aliquot C through ViveST
(elute Analyze plasma with ViveST (elute with Analyze plasma
samples with water) and analyze Abbott REALTIME water) and analyze
with with Abbott HCV GT HCV Assay with Abbott Abbott HCV GT Assay
Assay HCV Results LOG HCV Results LOG Results Genotype Results
Genotype Sample (IU/mL) (IU/mL) Interpretation Interpretation 1
3.92 3.66 2 2 2 5.46 5.32 1, 1a 1, 1a 3 4.58 4.24 1, 1a 1, 1a 4 3.6
3.34 1, 1a 1, 1a 5 4.62 4.14 1, 1a 1, 1a 6 4.7 3.92 1, 1a 1, 1a 7
5.39 5.02 1, 1a 1, 1a 8 4.85 4.59 1, 1a 1, 1a 9 4.98 4.69 1, 1a 1,
1a 10 2.96 2.8 2 2 11 6.03 5.74 3 3 12 5.44 5.15 1, 1a 1, 1a 13
4.48 4.02 3 3 14 5.07 4.77 3 3 15 3.46 3.4 1, 1a 1, 1a 16 5.32 4.94
1, 1a 1, 1a 17 5.47 5.26 1, 1b 1, 1b 18 2.91 2.64 1, 1a 1, 1a 19
4.89 4.48 1 1 Neg -- Not Detected -- Not Detected Control
TABLE-US-00005 TABLE 4 Results of Comparative Analysis Using the
Abbott's REALTIME HCV Testing (Fresh Plasma versus Samples
Processed through the ViveST Device of the Invention) Fresh Plasma,
Mean Viral Load LOG IU/mL (n = 19) 4.64 ViveST, Mean Viral Load LOG
IU/mL (n = 19) 4.32 Mean Difference, LOG IU/mL (Fresh vs. ViveST)
-0.32 Std. Dev. LOG IU/mL 0.15 Correlation Cofficient (R) 0.98
4. Conclusion
[0185] HCV genotyping results demonstrated 100% concordance between
the plasma samples recovered from the ViveST devices of the
invention as compared to the frozen plasma with HCV genotypes 1,
1a, 1b, 2, and 3 being tested (Table 3). HCV viral load results
showed an average reduction of 0.32 log for the plasma removed from
the ViveST devices of the invention as compared to the frozen
plasma (Table 4 and FIG. 9).
[0186] Based on dried blood/plasma spot data previously published,
one expects approximately 0.5-0.7 log reduction between
quantitation from fresh plasma versus a dried collection device
(Amellal et al., 2007, HIV Med. 8:396-400, discussing the median
loss was significant and equivalent to 0.64 log copies/mL; Hamers
et al., 2009, Antiviral Therapy 14:619-29, discussing the median
difference between DPS and plasma were 0.077 to 0.64 log
copies/mL). Here, this study demonstrated that the average viral
RNA recovered from the plasma samples processed through the ViveST
devices of the invention with the polyolefin matrix was surprising
higher and more reproducible than previously expected values based
on published literature.
Example 7
[0187] HIV-1 and ViroSeq HIV-1 Genotyping Evaluation for Samples
Processed through the ViveST Devices of the Invention
1. Experimental Design
[0188] 1 ml aliquots of HIV-1 positive plasma samples (N=20),
stored at -80.degree. C., was used for each part of the analysis
and was designated as follows:
[0189] A=sample analyzed with the Abbott REALTIME HIV-1 assay;
[0190] B=sample processed through the ViveST devices of the
invention with the polyolefin fiber matrix (elute with water) and
analyzed with the Abbott REALTIME HIV-1 assay;
[0191] C=sample processed through the ViveST devices of the
invention with the polyolefin fiber matrix (elute with mLysis
buffer) and analyzed with the Abbott REALTIME HIV-1 assay;
[0192] D=sample analyzed with the ViroSeq HIV-1 Pro & RT
Genotypic assay;
[0193] E=sample processed through the ViveST devices of the
invention with the polyolefin fiber matrix (elute with water) and
analyzed with the ViroSeq HIV-1 Pro & RT Genotypic assay.
[0194] Additional aliquots of each plasma sample were maintained at
-80.degree. C. for additional testing.
2.1 Procedure--Sampled Processed Through the ViveST Devices of the
Invention
[0195] a) Removed plasma aliquots designated as "1B, 2B . . . 20B",
"1C, 2C . . . 20C" and "1E, 2E . . . 20E" from -80.degree. C.,
thawed at room temperature;
[0196] b) Vortexed each sample to ensure adequate mixing;
[0197] c) Obtained 60 ViveST devices of the invention with the
polyolefin fiber matrix and labeled the cap of each with the sample
designations (i.e., 1b-20b, 1c-20c, 1e-20e), obtained 2 additional
ViveST devices and labeled each as Negative Control;
[0198] d) Loaded 1 ViveST device for each sample, loaded 1 mL
normal (HIV-1 negative) human plasma on the ViveST devices labeled
as Negative Controls;
[0199] e) Dried the loaded matrix in the ViveST devices in a
laminar flow hood for at least 12 hours but not more than 36
hours;
[0200] f) Recovered samples "1b-20b", "1e-20e" and "Neg Ctrl-water"
from the dried ViveST devices using water, recovered samples
"1c-20c" and "NegCtrl-lysis" with lysis buffer; and
[0201] g) Froze recovered samples at -80.degree. C.
2.2. Procedure--Abbott m2000 REALTIME HIV-1 Assay
[0202] a) Removed plasma aliquots designated as "1A, 2A . . . 20A",
and the recovered aliquots from the ViveST devices as "1B, 2B . . .
20B", "1C . . . 2C . . . 20C", "Neg Ctrl-water" and "Neg
Ctrl-lysis" from -80.degree. C., thawed at room temperature;
and
[0203] b) Processed the samples following the Abbott REALTIME HIV-1
assay package insert.
2.3 Procedure--ViroSeq HIV-1 Pro & RT Genotypic Assay
[0204] a) Removed plasma aliquots designated as "1D, 2D . . . 20D"
and the recovered aliquots from the ViveST devices as "1E, 2E . . .
20E" from -80.degree. C., thawed at room temperature; and
[0205] b) Processed the samples following the ViroSeq HIV-1 Pro
& RT Genotypic assay package insert.
3. Results--A Summary of the Results are Provided in Tables 5 and 6
Below.
TABLE-US-00006 [0206] TABLE 5 Summary of Abbott REALTIME HIV-1 and
ViroSeq HIV-1 Genotyping Results Aliquot B Aliquot C Process plasma
Process plasma Aliquot E samples through samples through Aliquot D
Process plasma Aliquot A ViveST (elute with ViveST (elute with
Analyze plasma samples through Analyze plasma water) and analyze
lysis) and analyze samples with ViveST and analyze with Abbott with
Abbott with Abbott ViroSeq HIV-1 with ViroSeq HIV-1 RealTime
RealTime RealTime Pro & RT Pro & RT HIV-1 Assay HIV-1 Assay
HIV-1 Assay Genotypic Assay Genotypic Assay bioMONTR Sample HIV-1
Results HIV-1 Results HIV-1 Results Genotypic Results Genotypic
Results ID ID LOG (c/mL) LOG (c/mL) LOG (c/mL) (see attached
report) (see attached report) b1208 1 5.45 4.68 4.61 NRTI: M41L,
E44D, D67N, NRTI: M41L, E44D, D67N, L74V, V118I, M184V, L74V,
V118I, M184V, L210W, T215Y, K219N L210W, T215Y, K219N NNRTI: V108I,
Y181C, NNRTI: V108I, Y181C, Y181L Y181L PI: L10I, V32I, M46I, PI:
L10I, V32I, M46I, F53L, I54V, Q58E, A71V, F53L, I54V, Q58E, A71V,
V82A, L90M V82A, L90M b1210 2 3.46 2.84 3.11 NRTI: M41L, E44D,
D67N, NRTI: M41L, E44D, D67N, K70R, M184V, L210W, K70R, M184V,
L210W, T215Y, K219E T215Y, K219E PI: L10I, I54V, V82A PI: L10I,
I54V, V82A b1211 3 6.07 5.6 5.75 NRTI: M41L, T215Y NRTI: M41L,
T215Y b1213 4 5.09 4.61 5.05 NRTI: M41L, E44D, L47V, NRTI: M41L,
E44D, L47V, L210W, T215Y L210W, T215Y NNRTI: Y188L NNRTI: Y188L PI:
M46I, A71T, L90M PI: M46I, A71T, L90M b1214 5 3.54 3.15 3.07 NRTI:
M41L, E44D, D67N, NRTI: M41L, E44D, D67N, T69D, V118I, T69D, V118I,
PI: L90M PI: L90M b1215 6 3.16 2.64 2.97 NRTI: M184V No
amplification b1215 7 2.12 1.57 2.03 Not analyzed due to Not
analyzed due to (diluted) low viral load low viral load b1216 8
4.57 4.14 4.37 Deletion @ T69 Deletion @ T69 (no report) (no
report) b1217 9 2.87 2.54 3.41 No amplification No amplification
b1217 10 2.22 1.29 2.28 Not analyzed due to Not analyzed due to
(diluted) low viral load low viral load b1218 11 4.72 4.15 4.3
NNRTI: K103N NNRTI: K103N, K238T b1219 12 4.17 3.67 3.79 NRTI: T69D
NRTI: T69D NNRTI: K103N NNRTI: K103N b1220 13 4.44 3.84 4.05 NRTI:
V75I No mutations Identified b1221 14 3.24 2.67 2.88 NRTI: D67N,
M184V, NRTI: D67N, M184V, T215Y T215C/T215Y NNRTI: K101Q, K103R,
NNRTI: K101Q, K103R, V179D, Y181C, G190A V179D, Y181C, G190A PI:
L10F, M461V, I54L, PI: L10F, M461V, I54L, I84V, L90M I84V, L90M
b1221 15 2.32 1.5 1.92 Not analyzed due to Not analyzed due to
(diluted) low viral load low viral load b1222 16 4.18 3.65 3.78 PI:
A71V PI: A71V b1223 17 2.7 2.04 3.06 No amplification No
amplification b1224 19 3.83 3.26 3.54 No Mutations Identified No
Mutations Identified b1225 19 4.19 3.41 3.77 NRTI: D67N, L74V,
V118I, NRTI: D67N, L74V, V118I, T215F, K219Q T215F, K219Q NNRTI:
L100I, K103N NNRTI: L100I, K103N PI: L10I, G48V, I54V, PI: L10I,
G48V, I54V, A71V, V82A, L90M A71V, V82A, L90M b1227 20 2.47 1.84
1.83 No amplification No amplification *All results are provided
for RESEARCH USE ONLY and should not be used for diagnostic
purposes.
TABLE-US-00007 TABLE 6 Results of Comparative Analysis Using the
Abbott's REALTIME HIV-1 Testing (Fresh Plasma versus Samples
Processed through the ViveST Devices) Fresh Plasma, Mean LOG c/mL
3.74 (n = 20) Recovery with Recovery with mLysis Water ViveST, Mean
LOG c/mL (n = 20) 3.48 3.15 Mean Difference, LOG c/mL -0.26 -0.59
(Fresh vs ViveST) Std Dev, LOG c/mL 0.31 0.15 Correlation
Cofficient (R) 0.96 0.99
4. Conclusions
[0207] HIV-1 viral load results showed an average reduction of 0.26
log for the plasma samples recovered from the ViveST devices of the
invention with the polyolefin fiber matrix using mLysis buffer and
0.59 log reduction using water (FIG. 10, FIG. 11 and Table 6).
[0208] HIV-1 drug resistance mutations were identified in 10/17
pairs (59%) and demonstrated 100% concordance between the plasma
samples recovered from the ViveST devices as compared to the frozen
plasma. A mixture T215Y/C was identified in 1/17 in the sample
recovered from the ViveST device while corresponding plasma
reported a mutation T215Y. A mutation at V75I was identified for
1/17 in the plasma sample while the paired sample recovered from
the ViveST device was wild type. 1/17 pair demonstrated deletion at
T69 preventing generation of ViroSeq report. 1/17 plasma sample had
M184V but results were not generated for corresponding processed
sample through the ViveST device due to low viral load. No
genotypic results were generated for 3/17 paired samples due to low
viral loads (Table 5).
[0209] Based on dried blood/plasma spot data previously published,
one expects approximately 0.5-0.7 log reduction between
quantitation from fresh plasma versus a dried collection device
(Amellal et al., 2007, HIV Med. 8:396-400, discussing the median
loss was significant and equivalent to 0.64 log copies/mL; Hamers
et al., 2009, Antiviral Therapy 14:619-29, discussing the median
difference between DPS and plasma were 0.077 to 0.64 log
copies/mL). Additionally, based on published data, one expects not
to obtain sufficient integrity (quality and quantity) of amplicons
for valid genotypic analysis results especially from low viremia
HIV samples (Lofgren et al., 2009, AIDS 23: 2459-66, providing that
performance was best for samples from patients with plasma RNA
levels about 5,000 copies/mL for detecting virologic failure;
Hamers et al., 2009, Antiviral Therapy 14:619-29, providing that
overall amplification success rates high for high VL (>3.0 to
4.0 log copies/mL), but reduced for lower VL (<3.0 log, reduced
sensitivity due to small volume used in spot, compared to 140 mL
for plasma).
[0210] Here, this study demonstrated the average viral RNA
recovered from the ViveST device of the invention with the
polyolefin matrix was surprisingly higher (recovered 0.26 log) and
more reproducible than previously expected values based on
published literature. Additionally, results were obtained in 14/17
pairs analyzed (82%) across a broad viral RNA range, is a greater
genotyping success rate than expected.
Example 8
HCV Validation (Linearity and Precision) for Samples Processed
Through the ViveST Devices of the Invention
1. Experimental Design
[0211] The purpose of this study was to validate the analytical
measurement range and the precision for the samples processed
through the ViveST devices of the invention with the polyolefin
matrix using the Abbott REALTIME HCV assay. This example describes
the verification of the analytical measurement range (linear range)
and precision.
2. Precision (Inter and Intra-Assay Precision)
[0212] Three samples of varying viral load values (low, mid, and
high viral load) stored in ViveST devices of the invention were
tested in triplicate on three separate assays performed by two
different operators on different days (N=27 samples). Table 7
describes the nomenclature of the experimental design for the
precision validation assays.
3. Analytical Measurement Range
[0213] For testing analytical measurement range, a high titer
sample (4E6 IU/mL) was serially diluted in normal human plasma to
yield dilutions of 1:2, 1:20, 1:200, 1:2,000, 1:20,000 and
1:200,000, and processed through the ViveST devices of the
invention with the polyolefin fiber matrix. Each dilution is tested
in triplicate in a single run on the m2000 platform (N=21). Table 8
describes the nomenclature of the experimental design for the
analytical measurement range validation assays. Additional aliquots
of each plasma sample were maintained at -80.degree. C. for
additional testing.
4. Procedures
[0214] a) Samples for the precision assays were HCV positive
samples of known concentrations. Samples for the analytical
measurement range assay were prepared as follows: high titer
samples were diluted to make 6 serial dilutions resulting in seven
samples with a concentration range of 1.3-6.6 log 10 IU/mL. The
samples were prepared in triplicate as indicated in Table 8;
[0215] b) Vortexed each sample to ensure adequate mixing;
[0216] c) Obtained 51 ViveST devices with the polyolefin fiber
matrix and labeled the cap of each with the sample designations
(Table 7);
[0217] d) Loaded 1 ViveST device for each sample (1.0 mL each),
loaded 1 mL normal (HCV negative) human plasma on the ViveST
devices labeled as Negative Controls;
[0218] e) Dried the loaded matrixes in the ViveST devices in a
laminar flow hood for at least 12 hours but not more than 36
hours;
[0219] f) Recovered samples from the dried ViveST devices using
water; and
[0220] g) Processed the samples following the Abbott REALTIME HCV
assay package insert.
[0221] Results for the HCV precision assay are provided in Tables 7
and 9. Results for the HCV analytical measurement range
determination are provided in Table 8 and FIG. 12.
TABLE-US-00008 TABLE 7 HCV Precision Data using the Abbott REALTIME
HCV Assay ViveST Abbott HCV Reproducibility Data Summary HCV titre
results Assay Sample ID Level (log.sub.10 IU/mL) Day 1 RLAV1A Low
2.16 RLAV1B 2.18 RLAV1C 2.30 RMAV1A Med 3.67 RMAV1B 3.71 RMAV1C
3.74 RHAV1A High 5.15 RHAV1B 5.17 RHAV1C 5.18 Day 2 RLAV2A Low 2.15
RLAV2B 2.21 RLAV2C 2.18 RMAV2A Med 3.58 RMAV2B 3.58 RMAV2C 3.62
RHAV2A High 6.04 RHAV2B 6.08 RHAV2C 4.98 Day 3 RLAV3A Low 2.31
RLAV3B 2.23 RLAV3C 2.11 RMAV3A Med 3.61 RMAV3B 3.64 RMAV3C 3.85
RHAV3A High 5.08 RHAV3B 5.19 RHAV3C 5.11
TABLE-US-00009 TABLE 8 Data for HCV Analytical Measurement Range
Determination using the Abbott REALTIME HCV Assay Target
Concentration HCV titre HCV titre (Log.sub.10 Sample ID Dilution
Replicate (log.sub.10 IU/mL) IU/mL) LAV7A 1 A 1.3 0.87 LAV7B 1 B
0.86 LAV7C 1 C 0.27 LAV6A 2 A 2.3 1.78 LAV6B 2 B 1.78 LAV6C 2 C
1.61 LAV5A 3 A 3.3 2.73 LAV5B 3 B 2.73 LAV5C 3 C 2.73 LAV4A 4 A 4.3
3.74 LAV4B 4 B 3.64 LAV4C 4 C 3.74 LAV3A 5 A 5.3 4.77 LAV3B 5 B
4.71 LAV3C 5 C 4.78 LAV2A 6 A 6.3 5.84 LAV2B 6 B 5.82 LAV2C 6 C
5.87 LAV1A 7 A 6.6 6.32 LAV1B 7 B 6.23 LAV1C 7 C 6.14
TABLE-US-00010 TABLE 9 HCV Inter-assay and Intra-assay Precision
Determination using the Abbott REALTIME HCV Assay ViveST Abbott HCV
Intra-assay and Inter-assay Precision Intra-assay precision
Inter-assay precision Concentration: Low Medium High Timepoint
(Day) 1 2 3 1 2 3 1 2 3 Low Medium High Replicates (n) 3 3 3 3 3 3
3 3 3 9 9 9 Mean (log.sub.10 IU/mL) 2.21 2.18 2.22 3.71 3.63 3.63
5.16 5.03 5.10 2.20 3.66 5.10 Standard Deviation 0.08 0.03 0.10
0.04 0.05 0.02 0.01 0.05 0.02 0.07 0.05 0.06 Coefficent of
variation (% CV) 0.04 0.01 0.05 0.02 0.02 0.01 0.00 0.02 0.01 0.06
0.05 0.06
5. Conclusions
[0222] This study determined that the analytical measurement range
of HCV positive samples processed through the ViveST devices of the
invention is 201 U/mL-4,000,000 IU/mL or 1.3-6.6 log 10 IU/mL.
Linear regression analysis R.sup.2 value is 0.9979 for the
analytical measurement range samples. The standard deviation for
all precision assays was <.+-.0.2 log 10 IU/mL indicating robust
reproducibility. The coefficient of variation (% CV) at a 95%
confidence level for inter-assay precision was <0.06% for all
time points for all sample concentrations. The coefficient of
variation (% CV) at a 95% confidence level for intra-assay
precision was <0.05% for all time points for all sample
concentrations.
[0223] Previously published dried blood spot and dried plasma spot
data indicated at least 0.5 log. Standard Deviation for precision
leading to highly variable recovery and reduced reproducibility for
viral load analysis (Andreotti et al., 2010, Clin. Virol. 47: 4-7,
discussing that 10% cases (n=13) DBS RNA not detectable while
measurable in plasma (between 2.1 and 3.04 log). One DBS gave 2.74
log while corresponding plasma level was <1.67 log. In all other
cases, undetectable RNA in plasma was not detectable in DBS
(n=18)). Here, this study demonstrated surprising reproducibility
across a broad viral load range indicating very robust storage and
recovery of nucleic acid using the ViveST devices of the invention
with the polyolefin matrix.
Example 9
HCV Evaluation for Samples Processed through the ViveST Devices of
the Invention using the Roche TaqMan HCV Assay
1. Experimental Design
[0224] The purpose of this study was to evaluate HCV in samples
processed through the ViveST devices of the invention with the
polyolefin matrix using the Roche COBAS AmpliPrep/COBAS TaqMan HCV
assay. For analysis using the Roche COBAS AmpliPrep/COBAS TaqMan
HCV assay, 1.2 mL aliquots of HCV positive plasma samples (N=20),
were used for the analysis. Additional aliquots of each plasma
sample were maintained at -80.degree. C. for additional
testing.
2. Procedure
[0225] a) High titer samples were diluted in HCV negative normal
human plasma to generate samples as described in Table 10;
[0226] b) Vortexed each sample to ensure adequate mixing;
[0227] c) 1.2 mL of each sample was stored frozen at -80.degree. C.
(pending analysis) or processed through the ViveST devices of the
invention as indicated in Table 10;
[0228] d) Obtained 20 ViveST devices and labeled the cap of each
with the sample designations (Table 10);
[0229] e) Load 1 ViveST device for each sample (1.2 mL each);
[0230] f) Dried the loaded matrixes in the ViveST devices in a
laminar flow hood for at least 12 hours but not more than 36
hours;
[0231] g) Recovered samples from the ViveST devices using 1.2 mL
water; and
[0232] h) Froze the recovered samples at -80.degree. C.;
[0233] i) Recovered samples, and stored frozen plasma aliquots were
analyzed using the Roche COBAS AmpliPrep/COBAS TaqMan HCV
assay.
3. Results--Results are provided in Table 10 below.
TABLE-US-00011 TABLE 10 Samples/Results for the Roche COBAS
AmpliPrep/ COBAS TAQMAN HCV Assay Process plasma samples through
ViveST (elute with Analyze plasma with water) and analyze Roche
COBAS .RTM. with Roche COBAS .RTM. Difference (plasma
AmpliPrep/COBAS .RTM. AmpliPrep/COBAS .RTM. sample processed Target
TaqMan .RTM. HCV assay TaqMan .RTM. HCV assay through ViveST vs.
Concentration HCV Results HCV Results plasma sample) Sample ID
(log.sub.10 IU/mL) log.sub.10 IU/mL log.sub.10 IU/mL log.sub.10
IU/mL 1a 6.6 6.45 6.37 -0.08 1b 6.39 6.39 0.00 2a 6.3 6.11 6.06
-0.05 2b 6.13 6.00 -0.13 3a 5.3 4.97 4.83 -0.14 3b 4.93 4.85 -0.08
4a 4.3 4.12 3.89 -0.23 4b 4.05 3.75 -0.30 5a 3.3 3.12 2.87 -0.25 5b
3.13 2.85 -0.28 6a 2.3 2.08 1.05 -1.03 6b 2.16 1.48 -0.68 7a 1.3
<1.18 <1.18 ND 7c <1.18 <1.18 ND La 2.76 3.03 2.27
-0.76 Lb 2.91 2.62 -0.29 Ma 4.48 4.55 3.89 -0.88 Mb 4.51 3.95 -0.56
Ha 6.07 6.01 5.92 -0.09 Hb 5.97 5.90 -0.07 Mean Difference
(log.sub.10 IU/mL): -0.32 ND = Difference not determined as result
values were <15 IU/mL.
4. Conclusions
[0234] Average loss of HCV RNA observed when plasma was processed
through the ViveST devices of the invention in the Roche COBAS
AmpliPrep/COBAS TaqMan HCV assay is 0.32 log 10 IU/mL (Table 10).
The linearity when the plasma samples were compared with the plasma
samples processed through the ViveST devices of the invention over
the concentration range of 2.3-6.6 log 10 IU/mL had a linear
regression analysis value (R.sup.2) of 0.9874 (FIG. 13).
[0235] Based on dried blood/plasma spot data previously published,
one expects approximately 0.5-0.7 LOG reduction between
quantitation from fresh plasma versus a dried collection device
(Amellal et al., 2007, HIV Med. 8:396-400, discussing the median
loss was significant and equivalent to 0.64 log copies/mL; Hamers
et al., 2009, Antiviral Therapy 14:619-29, discussing the median
difference between DPS and plasma were 0.077 to 0.64 log
copies/mL). Here, this study demonstrated that the average viral
RNA recovered using the ViveST devices of the invention with the
polyolefin matrix was surprising higher and more reproducible than
previously expected values based on published literature.
Example 10
HIV Evaluation for Samples Processed through the ViveST Devices of
the Invention using the Roche HIV1 RNA TagMan Assay
1. Experimental Design
[0236] The purpose of this study was to evaluate HIV in the samples
processed through the ViveST devices of the invention using the
Roche HIV1 RNA TaqMan assay. For analysis using the Roche assay,
1.2 mL aliquots of HIV-1 positive plasma samples (N=20), stored at
-80.degree. C., were used and aliquots were designated as
follows:
[0237] p=plasma sample analyzed with Roche HIV-1 RNA TaqMan
assay
[0238] v=plasma sample processed through the ViveST devices with
polyolefin fiber matrix (elute with water) and analyzed with the
Roche HIV-1 RNA TaqMan assay.
[0239] Additional aliquots of each plasma sample were maintained at
-80.degree. C. for additional testing.
2. Procedures
[0240] a) Removed plasma aliquots to be processed through the
ViveST devices from -80.degree. C., thawed at room temperature;
[0241] b) Vortexed each sample to ensure adequate mixing;
[0242] c) Obtained 20 ViveST devices and labeled the cap of each
with the sample designations (Table 11);
[0243] d) Loaded 1 ViveST device for each sample (1.2 mL each);
[0244] e) Dried the loaded matrixes in the ViveST devices in a
laminar flow hood for at least 12 hours but not more than 36
hours;
[0245] f) Recovered samples from the ViveST devices using water
(1.2 mL each);
[0246] g) Froze recovered samples at -80.degree. C.; and
[0247] h) Recovered samples and stored frozen plasma aliquots were
analyzed using the Roche HIV-1 RNA TaqMan assay.
3. Results--Results are Provided in Table 11 Below and FIG. 14.
TABLE-US-00012 [0248] TABLE 11 Samples/Results for the Roche HIV-1
RNA TaqMan Assay Aliquot `p` Aliquot `v` Roche HIV Roche HIV
Difference Plasma Results ViveST Results (ViveST - Sample ID LOG
(c/mL) LOG (c/mL) Plasma) b1209-21 4.41 4.13 -0.28 b1210-2 3.51
3.09 -0.42 b1208-1 5.5 5.14 -0.36 b1212-23 2.58 2.34 -0.24 b1213-4
5.33 5.17 -0.16 b1214-5 3.63 3.48 -0.15 b1215-7 2.38 <1.68 ND
b1215-24 3.43 3.18 -0.25 b1216-8 4.51 4.45 -0.06 b1217-10 2.46 1.94
-0.52 b1218-11 4.87 4.59 -0.28 b1219-12 4.32 4.29 -0.03 b1220-13
4.45 4.42 -0.03 b1221-15 2.11 2.12 0.01 b1222-16 4.45 4.53 0.08
b1223-17 3.46 3.01 -0.45 b1224-18 4.24 4.15 -0.09 b1225-19 4.26
3.75 -0.51 b1226-22 5.02 4.9 -0.12 b1227-20 2.46 2.24 -0.22 AVERAGE
3.87 3.73 -0.21 ND = Difference not determined as results values
were <1.68 LOG c/mL.
4. Conclusions
[0249] Average loss of HIV RNA observed when the plasma sample was
processed through the ViveST devices in the Roche HIV-1 RNA TaqMan
assay is 0.21 log c/mL (Table 11). The linearity when the plasma
samples were compared with the plasma samples processed through the
ViveST devices over the concentration range of .about.2.1-5.5 log
c/mL has a linear regression analysis value (R.sup.2) of 0.9717
(FIG. 14).
[0250] Based on dried blood/plasma spot data previously published,
one expects approximately 0.5-0.7 log reduction between
quantitation from fresh plasma versus a dried collection device
(Amellal et al., 2007, HIV Med. 8:396-400, discussing the median
loss was significant and equivalent to 0.64 log copies/mL; Hamers
et al., 2009, Antiviral Therapy 14:619-29, discussing the median
difference between DPS and plasma were 0.077 to 0.64 log
copies/mL). Here, this study demonstrated that the average viral
RNA recovered using the ViveST devices of the invention with the
polyolefin matrix was surprising higher and more reproducible than
previously expected values based on published literature.
Example 11
HIV-1 Assay Validation (Linearity and Precision) for Samples
Processed through the ViveST Devices of the Invention
1. Experimental Design
[0251] The purpose of this study was to validate the analytical
measurement range and the precision for the samples processed
through the ViveST devices of the invention using the Abbott
REALTIME HIV-1 assay. This example describes the verification of
the analytical measurement range (linear range) and precision.
2. Precision (Inter and Intra-Assay Precision)
[0252] Three samples of varying viral load values (low, mid, and
high viral load) stored in the ViveST devices were tested in
triplicate (at a minimum) on three separate assays on different
days.
3. Analytical Measurement Range
[0253] For testing analytical measurement range, a high titer
sample (-8 log copies/mL) was serially diluted in normal human
plasma to yield dilutions of 1:10, 1:100, 1:1,000, 1:10,000,
1:100,000, 1:1,000,000, and 1:10,000,000 and processed through the
ViveST devices of the invention with the polyolefin fiber matrix.
Each dilution was tested in triplicate on a single run on the m2000
platform (N=21). Serial dilutions were frozen, thawed, and analyzed
on the m2000 platform (N=21) for comparison. Additional aliquots of
each plasma sample were maintained at -80.degree. C. for additional
testing.
4. Procedure
[0254] a) Samples for the precision assays were diluted from a
HIV-1 positive sample with concentration of .about.8 log copies/mL.
Serial dilutions were made with negative human plasma to yield
samples with concentrations of .about.5 log copies/mL, .about.4 log
copies/mL, and .about.3 log copies/mL. Samples for the analytical
measurement range assay were prepared as follows: a high titer
sample was serially diluted 7 times resulting in seven samples with
a concentration range of 1-7 log copies/mL. The samples were
prepared in triplicate;
[0255] b) Vortexed each sample to ensure adequate mixing;
[0256] c) Obtained the appropriate number of ViveST devices and
labeled the cap of each with the sample designations;
[0257] d) Loaded 1 ViveST device for each sample (1.15 mL each),
loaded 1.15 mL normal (HIV-1 negative) human plasma on the ViveST
devices labeled as Negative Controls. NOTE: The Abbott REALTIME
HIV-1 0.6 mL application requires 1.1 mL sample; therefore, 1.15 mL
sample was loaded/recovered on the ViveST devices to ensure
adequate recovery;
[0258] e) Dried the loaded matrixes in the ViveST devices in a
laminar flow hood for at least 12 hours but not more than 36 hours,
designated the load/recovery dates on the assay worksheet, once
dried, capped the ViveST devices and stored at ambient laboratory
conditions;
[0259] f) Recovered samples from the dried ViveST devices using
water (1.15 mL each); and
[0260] g) Processed the samples following the Abbott REALTIME HIV-1
package insert (0.6 mL application).
[0261] Results for the HIV-1 precision assay are provided in Table
12. Results for the HIV-1 analytical measurement range
determination are provided in Table 13, and FIG. 15.
TABLE-US-00013 TABLE 12 HIV-1 Inter-Assay and Intra-Assay Precision
Determination using the Abbott REALTIME HIV-1 Assay Abbott HIV-1
Intra-Assay and Inter-Assay Precision Intra-assay precision
Inter-assay precision Concentration: Low Medium High Days stored on
ViveST 7 10 1 7 10 1 7 10 1 Low Medium High Replicates (n) 5 5 5 5
5 5 5 5 5 15 15 15 Mean 2.71 2.56 2.66 3.64 3.56 3.60 4.72 4.61
4.62 2.65 3.61 4.65 Standard Deviation 0.06 0.08 0.11 0.06 0.03
0.06 0.03 0.09 0.05 0.10 0.06 0.07 % CV 0.08 0.10 0.14 0.07 0.04
0.08 0.03 0.11 0.06 0.07 0.04 0.05
TABLE-US-00014 TABLE 13 HIV-1 Analytical Measurement Range
Determination using the Abbott REALTIME HIV-1 Assay Samples
processed Frozen through Samples Target HIV- Actual HIV- Actual
HIV- 1 titre 1 titre 1 titre (log10 c/mL) Difference 1 <1.6 ND
Not Calculated <1.6 ND Not Calculated ND <1.6 Not Calculated
2 <1.6 1.88 Not Calculated 1.70 <1.6 Not Calculated <1.6
ND Not Calculated 3 2.32 2.81 -0.49 2.48 2.70 -0.22 2.10 2.75 -0.65
4 3.13 3.71 -0.58 3.32 3.70 -0.38 3.07 3.67 -0.60 5 4.13 4.80 -0.67
4.24 4.80 -0.56 4.00 4.76 -0.76 6 5.06 5.84 -0.78 5.24 5.80 -0.56
5.08 5.76 -0.68 7 6.10 6.74 -0.64 6.04 6.75 -0.71 6.11 6.79 -0.68
Average difference: -0.60 ND = Target Not Detected Not Calculated =
Difference could not be calculated due to Target not detected or
Result of <1.6 log c/mL. indicates data missing or illegible
when filed
5. Conclusions
[0262] All analysis was performed using the Abbott REALTIME HIV-1
assay (0.6 mL application). This study determined that the
analytical measurement range of HIV-1 positive samples processed
through the ViveST devices of the invention is 2 log copies/mL-7
log copies/mL or 100 copies/mL-10,000,000 copies/mL. Linear
regression analysis R2 value is 0.9944 for the analytical
measurement range samples. A mean loss of 0.60 was observed for the
HIV-1 samples processed through and recovered from the ViveST
devices when compared to the frozen HIV-1 samples analyzed using
the Abbott REALTIME HIV-1m2000 system.
[0263] For the precision analysis: The standard deviation for all
assays was <.+-.0.2 log copies/mL indicating robust
reproducibility. The coefficient of variation (% CV) at a 95%
confidence level for inter-assay precision was <0.07% for all
time points for all sample concentrations. The coefficient of
variation (% CV) at a 95% confidence level for intra-assay
precision was <0.14% for all time points for all sample
concentrations.
Example 12
7-Day Stability Studies for HCV Using the Abbott HCV Assay
1. Experimental Design
[0264] The purpose of this study was to assess the ViveST devices
of the invention with the polyolefin fiber matrix for storage of
HCV infectious samples at ambient conditions over a seven day
period. HCV infectious samples at four concentrations were added to
the ViveST devices of the invention on Day 0 and dried overnight.
The ViveST devices were then be sealed by capping and stored at
ambient conditions. Samples were recovered and analyzed with the
Abbott REALTIME HCV assay on Day 1 (4 replicates each level), Day
3, and Day 7 (5 replicates each level). As a control, one frozen
plasma sample of each level was analyzed on Day 1. Negative
controls were included with each time point. The assay design is
shown in Table 14.
TABLE-US-00015 TABLE 14 Assay Design for HCV 7-Day Stability
Studies at Ambient Conditions Time point HCV titre level Replicates
Day 1 1 4 2 4 3 4 4 4 0 1 Day 3 1 5 2 5 3 5 4 5 0 1 Day 7 1 5 2 5 3
5 4 5 0 1
2. Procedures
[0265] a) Prepared HCV positive samples Levels 1-4 by linear
dilution (Table 15), used HCV negative plasma for negative
controls;
[0266] b) Vortexed each sample to ensure adequate mixing;
[0267] c) Obtained 60 ViveST devices and labeled the cap of each
with the sample designations, obtained additional 3 ViveST devices
for negative controls as described in Table 14;
[0268] d) Loaded 1 ViveST device for each sample (1.0 mL each) and
1 mL negative human plasma onto each negative control;
[0269] e) Dried the loaded matrixed in the ViveST devices in a
laminar flow hood for at least 12 hours but not more than 36
hours;
[0270] f) Recovered samples from the ViveST devices using water;
and
[0271] g) Processed the samples following the Abbott REALTIME HCV
assay package insert.
[0272] Results for the HCV 7-day stability studies at ambient
conditions are provided in Tables 15-16 below and FIGS. 16-18.
TABLE-US-00016 TABLE 15 Raw Data for HCV 7-Day Stability Studies at
Ambient Conditions using the Abbott REALTIME HCV Assay Assay
parameters Target Target Results HCV titre concentration
concentration HCV titre HCV titre Time point level (IU/mL) (log
IU/mL) Replicates Nomenclature log IU/mL IU/mL Day 1 Frozen plasma
1 1-1-A 3.98 9525 1 500 3.70 4 1-1-B Error Error 1-1-C 3.60 4007
1-1-D 3.57 3683 1-1-E 3.55 3530 Frozen plasma 1 1-2-A 3.67 4712 2
2500 3.40 4 1-2-B 3.25 1771 1-2-C 3.27 1874 1-2-D 3.23 1710 1-2-E
3.22 1662 Frozen plasma 1 1-3-A 3.32 2068 3 1250 3.10 4 1-3-B 2.99
974 1-3-C 3.02 1045 1-3-D 3.07 1196 1-3-E 3.00 994 Frozen plasma 1
1-4-A 3.09 1226 4 625 2.80 4 1-4-B 2.82 656 1-4-C 2.63 424 1-4-D
2.59 390 1-4-E 2.71 509 0 = neg 0 0 1 1-NEG not detected Day 3 1
5000 3.70 5 3-1-A 3.48 3003 3-1-B 3.44 2740 3-1-C 3.47 2940 3-1-D
3.48 3024 3-1-E 3.45 2799 2 2500 3.40 5 3-2-A 3.20 1593 3-2-B 3.16
1434 3-2-C 3.16 1444 3-2-D 3.13 1336 3-2-E 3.20 1582 3 1250 3.10 5
3-3-A 2.81 647 3-3-B 2.86 724 3-3-C 2.86 719 3-3-D 2.90 794 3-3-E
2.87 735 4 625 2.80 5 3-4-A 2.66 455 3-4-B 2.62 421 3-4-C 2.55 351
3-4-D 2.55 353 3-4-E 2.63 430 0 = neg 0 0 1 3-NEG not detected Day
7 1 5000 3.70 5 7-1-A 3.46 2856 7-1-B 3.32 2082 7-1-C 3.43 2721
7-1-D 3.42 2627 7-1-E 3.40 2500 2 2500 3.40 5 7-2-A 3.15 1424 7-2-B
2.98 960 7-2-C 3.13 1346 7-2-D 3.15 1414 7-2-E 3.18 1526 3 1250
3.10 5 7-3-A 2.85 714 7-3-B 2.82 656 7-3-C 2.97 940 7-3-D 2.87 745
7-3-E 2.93 852 4 625 2.80 5 7-4-A 2.45 280 7-4-B 2.55 353 7-4-C
2.59 393 7-4-D 2.47 294 7-4-E 2.570 371 0 = neg 0 0 1 7-NEG not
detected
TABLE-US-00017 TABLE 16 Result Summary for the HCV 7-day Stability
Studies at Ambient Conditions Using the Abbott REALTIME HCV Assay
Mean Mean difference(%) Mean loss Mean loss (log Mean loss Target
HCV Mean HCV HCV titre Difference(%) VivtST from (%) ViveST IU/mL)
ViveST (log IU/mL) titre (log titre results Standard ViveST vs
Initial from initial from initial ViveST from Time point IU/mL)
(log IU/mL) Deviation % CV frozen plasma timepoint timepoint
timepoint frozen plasma Frozen 3.70 3.96 N/A, n = 1 Nominal Not
applicable Plasma 3.40 3.67 3.10 3.32 2.80 3.09 Day 1 3.70 3.57
0.03 0.04 90 1st time point -0.41 3.40 3.24 0.03 0.04 88 -0.43 3.10
3.02 0.04 0.05 91 -0.30 2.80 2.69 0.06 0.09 87 -0.40 Day 3 3.70
3.46 0.02 0.02 87 97 -3.1 -0.11 -0.52 3.40 3.17 0.03 0.04 86 98
-2.2 -0.07 -0.50 3.10 2.86 0.03 0.04 86 95 -5.3 -0.16 -0.46 2.80
2.6 0.05 0.06 84 97 -3.2 -0.09 -0.49 Day 7 3.70 3.41 0.05 0.07 86
95 -4.7 -0.17 -0.57 3.40 3.12 0.08 0.10 85 96 -3.8 -0.12 -0.55 3.10
2.689 0.06 0.08 87 96 -4.4 -0.13 -0.43 2.80 2.53 0.06 0.08 82 94
-6.0 -0.16 -0.56 Minimum 2.80 2.53 0.02 0.02 82 94 -6.0 -0.17 -0.57
Maximum 3.70 3.98 0.08 0.10 91 98 -2.2 -0.07 -0.43
3. Conclusions
[0273] A maximum loss of 0.57 log IU/mL was recorded (Table 16 and
FIG. 18) between the frozen plasma as compared to the plasma
samples stored on the ViveST devices of the invention over a seven
day period at ambient temperature. A linear fit (R.sup.2>0.99)
was retained over the course of the 7-day study as indicated by
linear regression analysis of each level across all time points
(FIG. 16): R.sup.2 value of 0.9942 for Day 1 samples; R.sup.2 value
of 0.9987 for Day 3 samples; and R.sup.2 value of 0.9924 for Day 7
samples. The remarkable result here is the fact that regardless of
the level of viral RNA loaded onto the polyolefin matrix in the
ViveST devices of the invention, a very reproducible, predicable,
and quantifiable loss was demonstrated over time with R.sup.2
values at each viral RNA level >0.99.
Example 13
21-Day Stability Studies for HCV with the Storage Condition
Comparison
1. Experimental Design
[0274] The purpose of this study was to assess the ViveST devices
of the invention for storage of HCV infectious samples at various
storage conditions over a 21-day period. HCV infectious samples at
four concentrations were added to the ViveST devices of the
invention on Day 0 and dried overnight. The ViveST devices were
then sealed by capping and moved stored at ambient conditions (lab
bench), 4.degree. C. (refrigerator), and 40.degree. C./75% RH
(microclimate chamber). Samples (5 replicates each level) were
recovered from the ViveST devices and analyzed with the Abbott
REALTIME HCV assay on Day 1, Day 3, Day 7, Day 10, Day 14, and Day
21. As a control, frozen plasma samples (5 replicates each level)
were analyzed on Day 1. Negative controls were included with each
time point. The assay design is shown in Table 17.
TABLE-US-00018 TABLE 17 Assay Design for HCV 21-Day Stability
Studies with Storage Condition Comparisons Storage Days in Level
Level Level Level Neg Condition Storage 1 2 3 4 Control Frozen 0 5
5 5 5 1 Ambient 1 5 5 5 5 1 3 5 5 5 5 1 7 5 5 5 5 1 10 5 5 5 5 1 14
5 5 5 5 1 21 5 5 5 5 1 EXTRA* 5 5 5 5 1 4.degree. C. 1 5 5 5 5 1 3
5 5 5 5 1 7 5 5 5 5 1 10 5 5 5 5 1 14 5 5 5 5 1 21 5 5 5 5 1 EXTRA*
5 5 5 5 1 MicroClimate 1 5 5 5 5 1 Chamber 3 5 5 5 5 1 (40 C./75%
RH) 7 5 5 5 5 1 10 5 5 5 5 1 14 5 5 5 5 1 21 5 5 5 5 1 EXTRA* 5 5 5
5 1 *= In the event of a run failure, one extra set of samples was
made for each storage condition. If not used, these samples were
stored for future analysis.
2. Procedure
[0275] a) Prepared HCV positive samples Levels 1-4 by diluting a
high titer HCV infectious plasma sample into HCV negative (normal)
human plasma (Table 17), use HCV negative plasma for negative
controls;
[0276] b) Vortexed each sample to ensure adequate mixing;
[0277] c) Obtained 441 ViveST devices of the invention and labeled
the cap of each with the sample designations, 21 of these ViveST
devices were used for negative controls as described in Table 17,
additional aliquots (5 for each level+1 negative control) were
stored at -80.degree. C. and tested concurrently with the Day 1
samples;
[0278] d) Loaded 1 ViveST device for each sample (1.0 mL each) and
1 mL negative human plasma onto each negative control;
[0279] e) Dried the loaded matrixes in the ViveST devices in a
laminar flow hood for at least 12 hours but not more than 36 hours,
designated the load/recovery dates/times on the assay
worksheet;
[0280] f) Recovered samples from the ViveST devices using water;
and
[0281] g) Processed the samples following the Abbott REALTIME HCV
assay package insert.
3. Results--Results are Provided in Table 18-Table 20 and FIG.
19-FIG. 25.
TABLE-US-00019 [0282] TABLE 18 Results Summary for the HCV 21-Day
Stability Study at Ambient Storage Condition using the Abbott
REALTIME HCV Assay Target HCV titre Mean Results - Ambient Storage
(Days) (log IU/mL) Frozen 1 3 7 10 14 21 4.67 4.64 4.42 4.39 4.21
4.27 4.18 4.16 4.38 4.38 4.09 4.09 3.95 3.91 3.87 3.93 4.07 4.07
3.75 3.63 3.66 3.61 3.60 3.60 3.77 3.77 3.39 3.41 3.33 3.38 3.35
3.30
TABLE-US-00020 TABLE 19 Results Summary for the HCV 21-Day
Stability Study at 4.degree. C. Storage Condition using the Abbott
REALTIME HCV Assay Target HCV titre Mean Results - 4.degree. C.
Storage (Days) (log IU/mL) Frozen 1 3 7 10 14 21 4.67 4.64 4.45
4.38 4.28 4.36 4.32 4.37 4.38 4.38 4.11 4.09 4.01 4.01 4.01 4.06
4.07 4.07 3.79 3.76 3.68 3.71 3.70 3.71 3.77 3.77 3.51 3.45 3.37
3.45 3.38 3.38
TABLE-US-00021 TABLE 20 Results Summary for the HCV 21-Day
Stability Study at 40.degree. C./75% RH Storage Condition using the
Abbott REALTIME HCV Assay Target Mean Results - MIcroClimate
Chamber Storage, HCV titre 40.degree. C./75% RH (Days) (log IU/mL)
Frozen 1 3 7 10 14 21 4.67 4.64 4.20 4.10 3.91 3.89 3.84 3.76 4.38
4.38 3.93 3.78 3.62 3.58 3.52 3.45 4.07 4.07 3.54 3.52 3.30 3.25
3.24 3.13 3.77 3.77 3.35 3.26 2.97 2.98 3.01 2.89
4. Conclusions
[0283] For samples stored at ambient temperature, a maximum loss of
0.51 LOG IU/mL (range -0.23 to -0.51 LOG IU/mL) was recorded
between the frozen plasma and the plasma samples stored on the
ViveST devices of the invention over a 21-day period. The Standard
Deviation across all levels/all test points ranged from 0.01 to
0.17. For samples stored at 4.degree. C., a maximum loss of 0.40
LOG IU/mL (range -0.20 to -0.40 LOG IU/mL) was recorded between the
frozen plasma and the plasma samples stored on the ViveST devices
of the invention over a 21-day period. The Standard Deviation
across all levels/all test points ranged from 0.02 to 0.09. For
samples stored in the microclimate chamber at 40.degree. C./75% RH,
a maximum loss of 0.93 LOG IU/mL (range -0.42 to -0.93 LOG IU/mL)
was recorded between the frozen plasma and the plasma samples
stored on the ViveST devices of the invention over a 21-day period.
The Standard Deviation across all levels/all test points ranged
from 0.01 to 0.11.
[0284] A linear fit (R.sup.2>0.98) was retained over the course
of the 21-day study as indicated by linear regression analysis
across all test points and all storage conditions (FIGS. 19, 21,
and 23). A summary of linear regression results for Day 21 analysis
is: R.sup.2 value of 0.9970 for ambient storage; R.sup.2 value of
0.9997 for 4.degree. C. storage; and R.sup.2 value of 0.9959 for
40.degree. C./75% RH storage.
Example 14
28-Day Stability Studies for HIV at Ambient Conditions using the
Abbott REALTIME Using HIV-1 Assay
1. Experimental Design
[0285] The purpose of this study was to assess the ViveST devices
of the invention for storage of HIV-1 infectious samples at ambient
conditions over at least a twenty-eight day period.
[0286] HIV-1 infectious samples at four concentrations were added
to the ViveST devices of the invention on Day 0 and dried
overnight. The ViveST devices were then sealed by capping and
stored at ambient conditions. Samples were recovered and analyzed
with the Abbott REALTIME HIV-1 assay on Days 1, 3, 7, 10, 14, 21,
and 28. Five replicates of four concentration levels of .about.3,
.about.4, .about.5, and .about.6 log copies/mL were analyzed at
each test point. Negative controls were included with each time
point. Additional aliquots of each plasma sample will be maintained
at -80.degree. C. for additional testing.
2. Procedures
[0287] a) Prepared HIV-1 positive samples Levels 1-4 by linear
dilution (Table 21), used HIV-1 negative plasma for negative
controls;
[0288] b) Vortexed each sample to ensure adequate mixing;
[0289] c) Obtained 140 ViveST devices of the invention and labeled
the cap of each with the sample designations, obtained an
additional 7 ViveST devices for negative controls;
[0290] d) Load 1 ViveST for each sample (1.15 mL each) and 1.15 mL
negative human plasma onto each negative control; the Abbott
REALTIME HIV-1 0.6 mL application requires 1.1 mL sample,
therefore, 1.15 mL sample was loaded/recovered on the ViveST
devices to ensure adequate recovery;
[0291] e) Dried the loaded matrixes in the ViveST devices in a
laminar flow hood for at least 12 hours but not more than 36 hours,
designated the load/recovery dates on the assay worksheet; once
dried, capped the ViveST devices and stored at ambient laboratory
conditions;
[0292] f) Recovered samples from the ViveST devices using water
(1.15 mL each); and
[0293] g) Processed the samples following the Abbott REALTIME HIV-1
assay package insert (0.6 mL application).
3. Results--Results are Provided in Table 21, Table 22, FIG. 26,
and FIG. 27.
TABLE-US-00022 [0294] TABLE 21 Mean HIV-1 Titers for HIV-1
Stability Studies at Ambient Conditions using the Abbott REALTIME
HIV-1 Assay Target HIV-1 titre Test points (Days Stored on the
ViveST Devices) (log c/mL) Nominal 3 7 10 14 21 28 3 2.74 1.75 1.77
1.81 1.72 1.86 1.87 4 3.73 2.71 2.56 2.66 2.66 2.71 2.71 5 4.80
3.64 3.58 3.60 3.53 3.67 3.70 6 5.80 4.72 4.61 4.62 4.62 4.66 4.63
Note: Results of storage on the ViveST device for 1 Day were not
presented. There was a fatal run error on the Abbott m2000sp
resulting in the loss of all samples and no results were
obtained.
TABLE-US-00023 TABLE 22 Result Summary for the HIV-1 Stability
Studies at Ambient Conditions using the Abbott REALTIME HIV-1 Assay
Target Mean HIV Mean loss HIV titre HIV titre (log c/mL) titre (log
results Standard ViveST from Test point c/mL) (log c/mL) Deviation
% CV frozen plasma Frozen 3.00 2.74 0.16 0.20 Not Plasma 4.00 3.73
0.04 0.05 applicable 5.00 4.80 0.03 0.03 6.00 5.80 0.05 0.06 Day 3
3.00 1.75 0.05 0.07* -0.99 4.00 2.71 0.06* 0.08 -1.02 5.00 3.64
0.06 0.07 -1.15 6.00 4.72 0.03 0.03 -1.08 Day 7 3.00 1.77 0.28
0.36* -0.97 4.00 2.56 0.08 0.10 -1.17 5.00 3.58* 0.03 0.04 -1.22*
6.00 4.61 0.09 0.11 -1.19 Day 10 3.00 1.81 0.13 0.16 -0.94* 4.00
2.66 0.11 0.14 -1.07 5.00 3.60 0.06 0.08 -1.20 6.00 4.62 0.05 0.06
-1.18 Day 14 3.00 1.72 0.10 0.14 -1.03* 4.00 2.66 0.13 0.16 -1.07
5.00 3.53 0.15 0.19 -1.27 6.00 4.62 0.05 0.07 -1.18 Day 21 3.00
1.86 0.17 0.21 -0.88 4.00 2.71 0.04 0.04 -1.02 5.00 3.67 0.05 0.06
-1.13 6.00 4.66 0.04 0.04 -1.14 Day 28 3.00 1.87 0.21 0.26 -0.87
4.00 2.71 0.06 0.07 -1.02 5.00 3.70 0.08 0.10 -1.10 6.00 4.63 0.06
0.07 -1.17 Minimum 3.00 1.72 0.03 0.03 -1.27 Maximum 6.00 5.80 0.28
0.36 -0.87 Mean Loss (log c/mL) ViveST Compared to -1.09 Frozen
Plasma *= Data previously reported incorrectly. Data has been
corrected here-in
4. Conclusions
[0295] All analysis was performed using the Abbott REALTIME HIV-1
assay (0.6 mL application). For the analytical measure range
testing, samples stored on the ViveST devices for 1 day resulted in
a mean loss of 0.60 log c/mL when compared to frozen samples
(Example 11, HIV-1 Validation (Linearity and Precision)).
[0296] A mean loss of 1.09 log c/mL (Range=-0.87 log c/mL-1.27 log
copies/mL) was recorded (Table 22) between the frozen plasma and
the plasma samples stored on the ViveST devices of the invention
over a 28-day period at ambient temperature. A linear fit
(R.sup.2>0.9963) was retained over the course of the 28 day
study as indicated by linear regression analysis of each level
across all time points (FIG. 26): R.sup.2 value of 0.9989 for Day 3
samples; R.sup.2 value of 0.9963 for Day 7 samples; R.sup.2 value
of 0.9984 for Day 10 samples; R.sup.2 value of 0.9979 for Day 14
samples; R.sup.2 value of 0.9988 for Day 21 samples; and R.sup.2
value of 0.999 for Day 28 samples.
Example 15
62-Day Stability Studies for HCV with the Storage Condition
Comparisons
1. Experimental Design
[0297] This study served to supplement the HCV 21-day stability
studies in Example 13 with additional data collected after 62 days
of storage. During the original study, one extra set of samples
were loaded onto the ViveST devices of the invention for each
storage condition. These samples were not utilised during the
original 21 day study; therefore, they remained stored at the
relevant storage condition and were analyzed after 62 days. The
purpose of this study was to assess the ViveST devices of the
invention for storage of HCV infectious samples at various storage
conditions over a 60+ day period.
[0298] HCV infectious samples at four concentrations were added to
the ViveST devices of the invention on Day 0 and dried overnight.
The ViveST devices were then sealed by capping and moved stored at
ambient conditions (lab bench), 4.degree. C. (refrigerator), and
40.degree. C./75% RH (microclimate chamber). Samples (5 replicates
each level) were recovered from the ViveST devices and analyzed
with the Abbott REALTIME HCV Assay on Day 1, Day 3, Day 7, Day 10,
Day 14, Day 21, and Day 62. As a control, frozen plasma samples (5
replicates each level) were analyzed on Day 1. Negative controls
were included with each time point. The assay design is shown in
Table 23.
TABLE-US-00024 TABLE 23 Assay Design for HCV 62-Day Stability
Studies with Storage Condition Comparisons Storage Days in Level
Level Level Level Neg Condition Storage 1 2 3 4 Control Frozen 0 5
5 5 5 1 Ambient 1 5 5 5 5 1 3 5 5 5 5 1 7 5 5 5 5 1 10 5 5 5 5 1 14
5 5 5 5 1 21 5 5 5 5 1 62 5 5 5 5 1 4.degree. C. 1 5 5 5 5 1 3 5 5
5 5 1 7 5 5 5 5 1 10 5 5 5 5 1 14 5 5 5 5 1 21 5 5 5 5 1 62 5 5 5 5
1 MicroClimate 1 5 5 5 5 1 Chamber 3 5 5 5 5 1 (40 C./75% RH) 7 5 5
5 5 1 10 5 5 5 5 1 14 5 5 5 5 1 21 5 5 5 5 1 62 5 5 5 5 1
2. Procedure
[0299] a) Prepared HCV positive samples Levels 1-4 by diluting a
high titer HCV infectious plasma sample into HCV negative (normal)
human plasma (Table 23), used HCV negative plasma for negative
controls;
[0300] b) Vortexed each sample to ensure adequate mixing;
[0301] c) Obtained 441 ViveST devices of the invention and labeled
the cap of each with the sample designations, 21 of these ViveST
devices were used for negative controls as described in Table 23,
additional aliquots (5 for each level+1 negative control) were
stored at -80.degree. C. and tested concurrently with the Day 1
samples;
[0302] d) Loaded 1 ViveST device for each sample (1.0 mL each) and
1 mL negative human plasma onto each negative control;
[0303] e) Dried the loaded matrixes in the ViveST devices in a
laminar flow hood for at least 12 hours but not more than 36 hours,
designated the load/recovery dates/times on the assay
worksheet;
[0304] f) Recovered samples from the ViveST devices using water;
and
[0305] g) Processed the samples following the Abbott REALTIME HCV
package insert.
3. Results--Results are Provided in Table 24-Table 26 and FIG.
28-FIG. 34.
TABLE-US-00025 [0306] TABLE 24 Results Summary for the HCV 62-Day
Stability Studies at Ambient Storage Condition using the Abbott
REALTIME HCV Assay Target HCV titre Mean Results - Ambient Storage
(log IU/mL) Frozen 1 3 7 10 14 21 62 4.67 4.64 4.42 4.39 4.21 4.27
4.18 4.16 4.06 4.38 4.38 4.09 4.09 3.95 3.91 3.87 3.93 3.80 4.07
4.07 3.75 3.63 3.66 3.61 3.60 3.60 3.58 3.77 3.77 3.39 3.41 3.33
3.38 3.35 3.30 3.24
TABLE-US-00026 TABLE 25 Results Summary for the HCV 62-Day
Stability Studies at 4.degree. C. Storage Condition using the
Abbott REALTIME HCV Assay Target HCV titre Mean Results - 4.degree.
C. Storage (log IU/mL) Frozen 1 3 7 10 14 21 62 4.67 4.64 4.45 4.38
4.28 4.36 4.32 4.37 4.31 4.38 4.38 4.11 4.09 4.01 4.01 4.01 4.06
4.02 4.07 4.07 3.79 3.76 3.68 3.71 3.70 3.71 3.64 3.77 3.77 3.51
3.45 3.37 3.45 3.38 3.38 3.36
TABLE-US-00027 TABLE 26 Results Summary for the HCV 62-Day
Stability Studies at 40.degree. C./75% RH Storage Condition using
the Abbott REALTIME HCV Assay Target Mean Results - MIcroClimate
Chamber Storage HCV titre (40.degree. C./75% RH) (log IU/mL) Frozen
1 3 7 10 14 21 62 4.67 4.64 4.20 4.10 3.91 3.89 3.84 3.76 3.32 4.38
4.38 3.93 3.78 3.62 3.58 3.52 3.45 3.01 4.07 4.07 3.54 3.52 3.30
3.25 3.24 3.13 2.72 3.77 3.77 3.35 3.26 2.97 2.98 3.01 2.89
2.45
4. Conclusions
[0307] For samples stored at ambient temperature, a maximum loss of
0.58 LOG IU/mL (range -0.23 to -0.58 LOG IU/mL) was recorded
between the frozen plasma and the plasma samples stored on the
ViveST devices of the invention over a 62-day period. The Standard
Deviation across all levels/all test points ranged from 0.01 to
0.17. For samples stored at 4.degree. C., a maximum loss of 0.43
LOG IU/mL (range -0.20 to -0.43 LOG IU/mL) was recorded between the
frozen plasma and the plasma samples stored on the ViveST devices
of the invention over a 62-day period. The Standard Deviation
across all levels/all test points ranged from 0.02 to 0.09. For
samples stored in the microclimate chamber at 40.degree. C./75% RH,
a maximum loss of 1.37 LOG IU/mL (range -0.42 to -1.37 LOG IU/mL)
was recorded between the frozen plasma and the plasma samples
stored on the ViveST devices of the invention over a 62-day period.
The Standard Deviation across all levels/all test points ranged
from 0.01 to 0.11. For samples stored in the microclimate chamber,
an average loss of 0.88 LOG IU/mL was recorded between the samples
stored on the ViveST devices of the invention for 1 day and samples
stored for 62 days.
[0308] A linear fit (R.sup.2>0.98) was retained over the course
of the 62-day study as indicated by linear regression analysis
across all test points and all storage conditions (FIGS. 28, 30,
and 32). A summary of linear regression results for Day 62 analysis
is: R.sup.2 value of 0.9918 for ambient storage; R.sup.2 value of
0.9977 for 4.degree. C. storage; and R.sup.2 value of 0.9979 for
40.degree. C./75% RH storage.
Example 16
62-Day Stability Studies for HIV with Storage Condition
Comparisons
1. Experimental Design
[0309] The purpose of this study was to assess the ViveST devices
of the invention for storage of HIV-1 infectious samples at various
storage conditions over a 62-day period.
[0310] HIV-1 infectious samples at four concentrations were added
to the ViveST devices of the invention on Day 0 and dried
overnight. The ViveST devices were then be sealed by capping and
moved stored at ambient conditions (lab bench), 4.degree. C.
(refrigerator), and 40.degree. C./75% RH (microclimate chamber).
Samples (5 replicates each level) were recovered from the ViveST
devices and analyzed with the Abbott REALTIME HIV-1 assay on Day 1,
Day 3, Day 7, Day 10, Day 14, Day 21, and Day 62. As a control,
frozen plasma samples (5 replicates each level) were analyzed on
Day 1. Negative controls were included with each time point. The
assay design is shown in Table 27.
TABLE-US-00028 TABLE 27 Assay Design for HIV-1 62-Day Stability
Studies with Storage Condition Comparisons Storage Days in Level
Level Level Level Neg Condition Storage 1 2 3 4 Control Frozen 0 5
5 5 5 1 Ambient 1 5 5 5 5 1 3 5 5 5 5 1 7 5 5 5 5 1 10 5 5 5 5 1 14
5 5 5 5 1 21 5 5 5 5 1 62 5 5 5 5 1 4.degree. C. 1 5 5 5 5 1 3 5 5
5 5 1 7 5 5 5 5 1 10 5 5 5 5 1 14 5 5 5 5 1 21 5 5 5 5 1 62 5 5 5 5
1 MicroClimate 1 5 5 5 5 1 Chamber 3 5 5 5 5 1 (40.degree. C./75%
RH) 7 5 5 5 5 1 10 5 5 5 5 1 14 5 5 5 5 1 21 5 5 5 5 1 62 5 5 5 5
1
2. Procedures
[0311] a) Prepared HIV-1 positive samples Levels 1-4 by diluting a
high titer HIV-1 infectious plasma sample into HIV-1 negative
(normal) human plasma (Table 27), used HIV-1 negative plasma for
negative controls;
[0312] b) Vortexed each sample to ensure adequate mixing;
[0313] c) Obtained 441 ViveST devices of the invention and labeled
the cap of each with the sample designations, 21 of these ViveST
devices were used for negative controls as described in Table 27,
additional aliquots (5 for each level+1 negative control) were
stored at -80.degree. C. and tested concurrently with the Day 1
samples;
[0314] d) Loaded 1 ViveST device for each sample (1.1 mL each) and
1.1 mL negative human plasma onto each negative control, the Abbott
REALTIME HIV-1 0.6 mL application requires 1.1 mL sample,
therefore, 1.15 mL sample was loaded on each ViveST device to
ensure adequate recovery volume;
[0315] e) Dried the loaded matrixes in the ViveST devices in a
laminar flow hood for at least 12 hours but not more than 36 hours,
designated the load/recovery dates/times on the assay
worksheet;
[0316] f) Recovered samples from the ViveST devices using water
(1.15 mL each); and
[0317] g) Processed the samples following the Abbott REALTIME HIV-1
assay package insert (0.6 mL application).
3. Results--Results are provided in Table 28-Table 30 and FIG.
35-FIG. 41.
TABLE-US-00029 TABLE 28 Results Summary for the HIV-1 62-Day
Stability Studies at Ambient Storage Condition using the Abbott
REALTIME HIV-1 Assay Target HIV-1 titre Mean Results - Ambient
Storage (log c/mL) Frozen 1 3 7 10 14 21 62 5.00 4.89 4.21 4.15
4.09 4.12 4.10 4.07 3.98 4.70 4.59 3.94 3.90 3.87 3.82 3.79 3.83
3.77 4.40 4.32 3.63 3.67 3.57 3.63 3.57 3.61 3.51 4.10 4.01 3.28
3.35 3.30 3.27 3.27 3.27 3.23
TABLE-US-00030 TABLE 29 Results Summary for the HIV-1 62-Day
Stability Studies at 4.degree. C. Storage Condition using the
Abbott REALTIME HIV-1 Assay Target HIV-1 titre Mean Results -
4.degree. C. Storage (log c/mL) Frozen 1 3 7 10 14 21 62 5.00 4.89
4.17 4.09 4.10 4.11 4.05 4.06 4.08 4.70 4.59 3.86 3.85 3.86 3.81
3.85 3.79 3.79 4.40 4.32 3.63 3.59 3.54 3.58 3.58 3.52 3.57 4.10
4.01 3.40 3.30 3.29 3.28 3.28 3.22 3.26
TABLE-US-00031 TABLE 30 Results Summary for the HIV-1 62-Day
Stability Studies at 40.degree. C./75% RH Storage Condition using
the Abbott REALTIME HIV-1 Assay Target Mean Results - MIcroClimate
Chamber Storage HIV-1 titre (40.degree. C./75% RH) (log c/mL)
Frozen 1 3 7 10 14 21 62 5.00 4.89 4.04 3.96 3.89 3.86 3.83 3.74
3.20 4.70 4.59 3.79 3.70 3.64 3.62 3.55 3.47 3.12 4.40 4.32 3.49
3.43 3.37 3.31 3.28 3.25 2.86 4.10 4.01 3.22 3.17 3.07 2.99 2.97
2.99 2.57
4. Conclusions
[0318] For samples stored at ambient temperature, a maximum loss of
0.91 LOG c/mL (range -0.65 to -0.91 LOG c/mL) was recorded between
the frozen plasma and the plasma samples stored on the ViveST
devices of the invention over a 62-day period. The Standard
Deviation across all levels/all test points ranged from 0.02 to
0.13. For samples stored at 4.degree. C., a maximum loss of 0.84
LOG c/mL (range -0.60 to -0.84 LOG c/mL) was recorded between the
frozen plasma and the plasma samples stored on the ViveST devices
over a 62-day period. The Standard Deviation across all levels/all
test points ranged from 0.02 to 0.07. For samples stored in the
microclimate chamber at 40.degree. C./75% RH, a maximum loss of
1.69 LOG c/mL (range -0.79 to -1.69 LOG c/mL) was recorded between
the frozen plasma the plasma samples stored on the ViveST devices
of the invention over a 62-day period. The Standard Deviation
across all levels/all test points ranged from 0.02 to 0.12. For
samples stored in the microclimate chamber, an average loss of 0.70
LOG c/mL was recorded between the samples stored on the ViveST
devices of the invention for 1 day and samples stored for 62
days.
[0319] A linear fit (R.sup.2>0.95) was retained over the course
of the 62-day study as indicated by linear regression analysis
across all test points and all storage conditions (FIGS. 35, 37,
and 39). A summary of linear regression results for Day 62 analysis
is: R.sup.2 value of 0.9953 for ambient storage; R.sup.2 value of
0.9961 for 4.degree. C. storage; and R.sup.2 value of 0.9555 for
40.degree. C./75% RH storage.
Example 17
HIV-1 Limited of Detection (LOD) Evaluation using the Abbott HCV
Assay
1. Experimental Design
[0320] The purpose of this experiment was to evaluate the limit of
detection (LOD) for the ViveST devices of the invention to store
HIV-1 infectious plasma samples at ambient conditions over a seven
day period.
[0321] HIV-1 infectious plasma samples at six linear concentrations
(n=14 samples each level) were added to the ViveST devices of the
invention (Day 0), placed in a laminar flow hood and dried
overnight. The ViveST devices were then be sealed by capping and
stored at ambient conditions for seven days. Samples were recovered
from the ViveST devices on Day 7 and analyzed with the Abbott
REALTIME HIV-1 assay. An additional aliquot of each concentration
were stored frozen and analyzed concurrently with the recovered
samples from the ViveST devices. The assay design is shown in Table
31. Additional aliquots of each plasma sample were maintained at
-80.degree. C. for additional testing. All lot numbers were
recorded on the assay worksheet.
TABLE-US-00032 TABLE 31 Assay Design for HIV-1 LOD Studies HIV-1
Evaluation: Limit of Detection (LOD) - Abbott HCV Assay Target
Concentration Target Concentration Level (c/mL) (LOG c/mL)
Replicates 5 774 2.89 1 frozen 14 ViveST 6 387 2.59 1 frozen 14
ViveST 7 193 2.29 1 frozen 14 ViveST 8 97 1.99 1 frozen 14 ViveST 9
48 1.68 1 frozen 14 ViveST 10 24 1.38 1 frozen 14 ViveST
2.1 Procedures--Sample Processed Through the ViveST Devices of the
Invention
[0322] a) Samples for the LOD assay were diluted from a HIV-1
positive sample with concentration of .about.8 LOG c/mL, serial
dilutions were made with negative human plasma to yield samples
with concentrations are described in Table 31, approximately 20 mL
of each concentration was required;
[0323] b) Vortexed each sample concentration to ensure adequate
mixing;
[0324] c) Obtained 84 ViveST devices of the invention and labeled
the cap of each;
[0325] d) Loaded 14 ViveST devices for each sample concentration
(1.15 mL each); the Abbott REALTIME HIV-1 0.6 mL application
requires 1.1 mL sample, therefore, 1.15 mL sample was loaded on
each ViveST device to ensure adequate recovery volume;
[0326] e) Pipetted 1.1 mL of each sample concentration into a
sterile screw cap tube and stored at -80.degree. C.;
[0327] f) Dried the loaded matrixes in the ViveST devices in a
laminar flow hood for at least 12 hours but not more than 36 hours,
designated the load/recovery dates/times on the assay
worksheet;
[0328] g) Capped and sealed the loaded ViveST devices and stored at
ambient laboratory conditions for 7 days; and
[0329] h) Recovered samples from the ViveST devices using water
(1.15 mL each).
2.2 Procedure--Abbott m2000 REALTIME HCV Assay: Processed the
frozen samples and the recovered samples from the ViveST devices
following the Abbott REALTIME HIV-1 assay package insert (0.6 mL
application). 3. Results--Results for the HIV-1 infectious frozen
plasma samples not processed through the ViveST devices are
presented in Table 32 and FIG. 42. Results of the HIV-1 infectious
samples processed and recovered after storage on the ViveST devices
for 7 days are presented in Table 33, Table 34 and FIG. 43.
TABLE-US-00033 TABLE 32 HIV-1 LOD Studies for Frozen Plasma Samples
Not Processed Through the ViveST Devices Target Achieved Achieved
titre Target titre HIV-1 titre HIV-1 titre Level (c/mL) (LOG c/mL)
Sample ID (LOG c/mL) (c/mL) 10 24 1.38 frozen 10 TND TND 9 48 1.68
frozen 9 1.36 23 8 97 1.99 frozen 8 1.92 83 7 193 2.29 frozen 7
1.94 86 6 387 2.59 frozen 6 2.18 150 5 774 2.89 frozen 5 2.70
502
TABLE-US-00034 TABLE 33 Summary of the HIV-1 LOD Data (LOG c/mL)
using the Abbott REALTIME HIV-1 Assay Frozen Calculated Sample
Percent Mean Viral Load Number Number Detected Viral Load Level
(LOG c/mL) tested Detected (%) (LOG c/mL) 5 2.70 14 14 100% 1.82 6
2.18 14 10 71% 1.55 7 1.94 14 8 57% 1.36 8 1.92 14 7 50% 1.35 9
1.36 14 4 29% 1.20 10 TND 14 0 0% N/A
TABLE-US-00035 TABLE 34 Summary of the HIV-1 LOD Data (c/mL) using
the Abbott REALTIME HIV-1 Assay Frozen Calculated Sample Percent
Mean Viral Load Number Number Detected Viral Load Level (c/mL)
tested Detected (%) (c/mL) 5 502 14 14 100% 74 6 150 14 10 71% 42 7
86 14 8 57% 24 8 83 14 7 50% 27 9 23 14 4 29% 16 10 TND 14 0 0%
N/A
4. Conclusions
[0330] A high titer HIV-1 positive sample was diluted in normal
human plasma to yield dilutions of 6 concentrations. The diluted
samples yielded slightly lower values than expected; however,
linear regression analysis yielded an R.sup.2 value of 0.92067,
indicating the diluted samples were acceptable for use in this
study (Table 32 & FIG. 42). A maximum loss of 0.88 LOG c/mL of
HIV-1 RNA was observed for the plasma samples stored on the ViveST
devices for 7 days when compared to the frozen plasma (Table 33
& Table 34). Based on the Probit analysis of the data, when the
plasma sample with a HIV-1 concentration of 353 c/mL (2.55 LOG
c/mL), was loaded on the ViveST device and stored (up to 7 days),
that sample was detected with 95% probability (FIG. 43). For all
analysis, results reported as <40 c/mL (<1.60 LOG c/mL) by
the Abbott data analysis software were manually calculated using a
stored HIV-1 calibration curve.
Example 18
Evaluation of Samples Processed through the ViveST Devices for Use
in the Vitro Seq HIV-1 Genotyping System (v2.0)
1. Purpose
[0331] The purpose of this study was to evaluate the samples
processed through the ViveST devices of the invention for use in
the ViroSeq HIV-1 Genotyping System (v2.0). This example describes
the results of accuracy as compared to the frozen plasma not
processed through the ViveST devices.
2. Methodology
[0332] The ViroSeq HIV-1 Genotyping System (v2.0) is a qualitative
RNA-based cycle sequencing assay that detects HIV-1 genomic
mutations. The assay detects mutations in the entire protease
region and two-thirds of the reverse transcriptase region of the
HIV-1 pol gene. The assay is based on five major processes: reverse
transcription (RT); polymerase chain reaction (PCR); cycle
sequencing; automated sequence detection; and software
analysis.
[0333] The protease and reverse transcriptase regions were
amplified to generate a 1.8 kb amplicon. The amplicon were used as
a sequencing template for seven primers that generate an
approximately 1.3 kb consensus sequence. The ViroSeq HIV-1
Genotyping System (v2.8) software was used to compare the consensus
sequence with the known HXB-2 reference sequence to determine
mutations present in the sample.
3. Experimental Design
[0334] The performance of the samples processed through the ViveST
devices of the invention in the ViroSeq HIV-1 Genotyping System
(v2.0) was evaluated for accuracy as compared to the frozen plasma.
Comparative genotypic analysis was performed on duplicate aliquots
of ten (10) paired HIV-1 plasma samples (frozen vs. samples
processed through the ViveST devices) with viral loads ranging from
3.58 to 5.17 LOG c/ml. To assess reproducibility, of the ten paired
samples, replicates (neat, 1:2, and 1:4 dilutions) of two samples
and replicates (neat and 1:4 dilution) of one sample were analyzed.
Frozen plasma samples were extracted via EtOH (manual extraction
per the FDA approved package insert). The plasma samples processed
through the ViveST devices of the invention were extracted per
bioMONTR's research method (RM-005.00, Sequencing of HIV-1 Pro/RT
Region Using ViroSeq HIV-1 Genotyping System and the ABI Prism
3100/3130 Genetic Analyzer). This method utilises an automated RNA
extraction, paramagnetic silica particles using NucliSENS easyMag
platform (bioMerieux, Inc.). All HIV-1 sequencing reactions were
processed on an ABI PRISM 3100 Genetic Analyzer capillary platform
(Applied Biosystems) and data was analyzed using ViroSeq software
(v2.8). HIV-1 sequence homology was analyzed via bioMONTR's
proprietary bioConT sequence analysis tool.
4. Results
[0335] Drug resistance mutations were 100% concordant (10/10 pairs)
in ViroSeq HIV-1 generated reports between the plasma samples
processed through the ViveST devices of the invention and the
frozen plasma. HIV-1 drug resistance mutations were identified in
4/10 pairs with WT virus detected in 6/10 paired specimens. For all
of the paired samples, there was >99% concordance at the
nucleotide level when comparing the plasma samples processed
through the ViveST devices with the frozen plasma for the protease
and reverse transcriptase regions (Table 35). For the replicate
samples (neat, 1:2 and 1:4 dilutions), the plasma samples processed
through the ViveST devices of the invention produced the identical
drug resistance profile pattern regardless of the dilution
analyzed.
TABLE-US-00036 TABLE 35 Results of ViroSeq Analysis Concor- dance
NanoDrop based Concor- Sample Information Values on Drug dance
Dilu- purified Resis- at the tion Viral Load PCR tance Nucle- Sam-
Lev- Repli- Fac- LOG product Drug Resistance Mutations Muta- otide
ple el cate Assay tor c/mL c/mL (ng/ul) NRTI NNRTI PI tions Level 1
1 1 ETOH ViroSeq 1 148,140 5.17 19 No Mutations Identified 100%
99.92% 4 ViveST_easyMAG 14.1 No Mutations Identified 2 1 ETOH
ViroSeq 1:2 74,080 4.87 32.1 No Mutations Identified 100% 100.00% 2
ViveST_easyMAG 12.7 No Mutations Identified 3 1 ETOH ViroSeq 1:4
37,040 4.57 24.3 No Mutations Identified 100% 99.92% 2
ViveST_easyMAG 10 No Mutations Identified 2 1 1 ETOH ViroSeq 1
134,424 5.13 18.3 No Mutations Identified 100% 99.08% 2 ViveST
easyMAG 1.7 No Mutations Identified 2 1 ETOH ViroSeq 1:2 67,212
4.83 48.8 No Mutations Identified 100% 99.62% 2 ViveST_easyMAG 9.1
No Mutations Identified 3 1 ETOH ViroSeq 1:4 33,606 4.53 18.5 No
Mutations Identified 100% 99.54% 2 ViveST_easyMAG 7.3 No Mutations
Identified 3 1 1 ETOH ViroSeq 1 15,176 4.18 11.6 M41L, E44D, V108I,
L10I, V32I, 100% 99.46% D67N, L74I, Y181I M46I, F53L, L74V, V118I,
I54V, Q58E, M184V, L210W, A71V, V82A, T215Y, K219N L90M 2
ViveST_easyMAG 10.9 M41L, E44D, V108I, L10I, V32I, D67N, L74I,
Y181I M46I, F53L, L74V, V118I, I54V, Q58E, M184V, L210W, A71V,
V82A, T215Y, K219N L90M 3 1 ETOH ViroSeq 1:4 3,794 3.58 15.7 M41L,
E44D, V108I, L10I, V32I, 100% 99.23% D67N, L74I, Y181I M46I, F53L,
L74V, V118I, I54V, Q58E, M184V, L210W, A71V, V82A, T215Y, K219N
L90M 2 ViveST_easyMAG 7.8 M41L, E44D, V108I, L10I, V32I, D67N,
L74I, Y181I M46I, F53L, L74V, V118I, I54V, Q58E, M184V, L210W,
A71V, V82A, T215Y, K219N L90M 4 2 1 ETOH ViroSeq 1:2 28,400 4.45
4.1 M41L, T215Y 100% 99.00% 2 ViveST_easyMAG 4.1 M41L, T215Y 5 1 1
ETOH ViroSeq 1 24,336 4.39 7.1 M41L, T69N, K103N, L10F, V11I, 100%
99.54% K70R, M184V, V108I, K43T, I54V, L210W, T215F, Y181C A71V,
V82A, K219E I84V, L90M 2 ViveST_easyMAG 14.8 M41L, T69N, K103N,
L10F, V11I, K70R, M184V, V108I, K43T, I54V, L210W, T215F, Y181C
A71V, V82A, K219E I84V, L90M
5. Conclusions
[0336] The use of the samples processed through the ViveST devices
of the invention in the ViroSeq HIV-1 Genotyping System (v2.0)
demonstrated 100% concordance for drug resistance mutations and
greater than 99% at the nucleotide level as compared to the frozen
plasma. Additionally, replicates of plasma samples processed
through the ViveST devices of the invention generated identical
drug resistance mutation patterns. The results demonstrate the
ViveST devices' utility for transporting plasma obtained from HIV-1
positive individuals for HIV-1 resistance testing.
Example 19
The High Pure System Validation Using the Roche TagMan HCV
Assay
1. Purpose
[0337] The purpose of this study was to validate the ViveST devices
of the invention for use with The High Pure System using the Roche
COBAS TaqMan HCV (v 2.0) assay. This study describes the results
of: precision studies; linearity (analytical measurement range);
stability (7 days); accuracy as compared to the frozen plasma; and
limit of detection (LOD)/limit of quantitation (LOQ).
2. Methodology
[0338] The Roche COBAS TaqMan HCV (v2.0) for use with The High Pure
System is a quantitative RT-PCR based assay that uses RT-PCR to
generate amplified product from the RNA genome of HCV in clinical
specimens. The process is based on two major steps: a) extraction
of viral RNA from plasma samples, and b) amplification with
concurrent detection of viral RNA.
3. Experimental Design
[0339] All testing on the Roche COBAS TaqMan HCV (v2.0) for use
with The High Pure System was performed according to FDA approved
protocol (0.5 mL) with no modifications. The Roche HCV assay
requires 0.5 mL sample, therefore, 0.8 mL sample was loaded
on/recovered from each ViveST device to ensure adequate sample
volume. All loaded ViveST devices were stored at ambient
temperature (RT). The performance of the samples processed through
the ViveST devices of the invention in this assay was evaluated for
precision, accuracy, analytical measurement range, stability, and
limit of detection (LOD)/limit of quantitation (LOQ).
3.1 Precision (Inter and Intra-Assay Precision)
[0340] To assess inter- and intra-assay precision, HCV infectious
plasma samples with varying viral load values (low, mid, and high
viral load) were stored in triplicate on the ViveST devices of the
invention, recovered, and tested with the Roche HCV assay on
different days (n=27). A summary of the results is provided in
Table 36.
TABLE-US-00037 TABLE 36 Summary of Intra-Assay and Inter-assay
Precision (Mean Values) ViveST_Roche HCV Intra-assay and
Inter-assay Precision Intra-Assay Precision Inter-Assay Precision
Concentration Low Medium High Run # 1 2 3 1 2 3 1 2 3 Days Stored 1
3 7 1 3 7 1 3 7 Low Medium High Replicates 3 3 3 3 3 3 3 3 3 9 9 9
Mean 3.65 3.59 3.48 4.17 4.22 4.06 4.48 4.49 4.38 3.57 4.15 4.45
Std Dev 0.15 0.04 0.07 0.13 0.04 0.07 0.01 0.03 0.09 0.11 0.11 0.07
95% CI 0.17 0.04 0.08 0.15 0.04 0.08 0.01 0.03 0.10 0.07 0.07
0.04
Conclusion:
[0341] The inter-assay and intra-assay standard deviations (SDs)
achieved at mean concentrations of .about.3.55, .about.4.15 and
.about.4.45 LOG IU/mL are <0.15 log IU/mL indicating robust
reproducibility. The 95% confidence interval (95% CI) for
inter-assay precision was +/-0.07 for all time points for all
sample concentrations. The 95% confidence interval (95% CI) for
intra-assay precision was +/-0.17 for all time points for all
sample concentrations.
3.2 Analytical Measurement Range and Accuracy
[0342] For testing analytical measurement range, a high titer HCV
infectious plasma sample (.about.6 log copies/mL) was serially
diluted in normal human plasma (7 levels). Each level was loaded
onto the ViveST devices of the invention in triplicate, stored for
7 days, recovered, and tested on a single run (n=21). For accuracy
as compared to the frozen plasma, identical serial dilutions were
frozen (in triplicate), thawed, and analyzed (N=21). Results are
provided in Table 37 and FIG. 44.
TABLE-US-00038 TABLE 37 HCV Linearity Using the Roche COBAS TaqMan
HCV Test (v2.0) Actual Results ViveST Frozen ViveST Results
Standard Samples Results Average Deviation Difference Sample (LOG
(LOG (LOG (LOG Frozen Vs ID IU/mL) IU/mL) IU/mL) IU/mL) ViveST
Level 7 3.92 3.46 3.55 0.10 -0.37 3.55 3.65 Level 6 4.28 3.76 3.78
0.02 -0.50 3.79 3.78 Level 5 4.59 4.06 4.11 0.05 -0.48 4.14 4.14
Level 4 5.01 4.34 4.40 0.07 -0.61 4.37 4.48 Level 3 5.36 4.82 4.76
0.11 -0.60 4.83 4.64 Level 2 5.70 5.09 5.16 0.08 -0.54 5.14 5.25
Level 1 6.08 5.43 5.46 0.04 -0.62 5.44 5.51 Minimum Loss -0.37
Maximum Loss -0.62 Average Loss -0.53 (7 day storage)
Conclusion
[0343] This study confirmed good sample correlation across a range
of .about.3 to .about.6 LOG (samples processed through the ViveST
devices as compared to the frozen plasma) with linear regression
analysis yielding an R.sup.2 value of 0.9954. An average reduction
of 0.53 LOG IU/mL HCV RNA was observed for samples stored on the
ViveST devices for 7 days at ambient condition (RT) prior to
recovery and analysis when compared to the frozen plasma.
3.3 Stability
[0344] To assess stability, HCV infectious plasma samples with
varying viral load values (low, mid, and high viral load) were
analyzed on the Roche HCV assay after being stored at ambient
condition (RT) on the ViveST devices for 1, 3, and 7 days (n=27).
Results are provided in Table 38, FIG. 45 and FIG. 46.
TABLE-US-00039 TABLE 38 Results Summary for the HCV 7-Day Stability
Studies at Ambient Storage Conditions using the Roche COBAS TaqMan
HCV Test (v2.0) Mean Results - Target HCV titre Ambient Storage
(LOG IU/mL) Frozen 1 3 7 4.70 4.94 4.48 4.49 4.38 4.30 4.63 4.17
4.22 4.06 3.78 3.96 3.65 3.59 3.48
Conclusion
[0345] For samples stored at ambient temperature (RT), a maximum
reduction of 0.57 LOG IU/mL (range 0.31 to 0.57 LOG IU/mL) was
recorded between the frozen plasma and the plasma samples stored on
the ViveST devices of the invention over a 7-day period (Table 38).
The Standard Deviation across all levels/all test points ranged
from 0.01 to 0.15. A linear fit (R.sup.2>0.97) was retained over
the course of the 7-day study as indicated by linear regression
analysis across all time points (FIG. 45).
3.4 Limit of Detection (LOD)/Limit of Quantitation (LOQ)
[0346] For determination of the LOD/LOQ, HCV infectious plasma was
diluted in HCV negative human plasma to yield dilutions of
approximately 40 to 440 IU/mL. To confirm the HCV RNA
concentration, the diluted samples were analyzed and linear
regression analysis was performed. 20 replicates of each
concentration were then loaded onto the ViveST devices of the
invention and stored for 7 days at ambient condition (RT). After
recovery, samples were tested using a single lot of extraction and
amplification reagents. The Probit analysis was performed to
determine the 95% hit rate.
TABLE-US-00040 TABLE 39 HCV LOD/LOQ Studies for Frozen Plasma
Samples Not Processed through ViveST using the Roche COBAS TaqMan
HCV Assay Achieved Target Achieved HCV Target titre HCV Titre titre
(LOG Sample titer (LOG Level (IU/mL) IU/mL) ID (IU/mL) IU/mL) 6
12.5 1.10 frozen 6 37 1.55 5 25 1.40 frozen 5 72 1.85 4 50 1.70
frozen 4 142 2.14 3 75 1.88 frozen 3 178* 2.25* 2 100 2.00 frozen 2
215 2.32 1 200 2.30 frozen 1 436 2.64 *= Error during analysis.
Value estimated using values of Level 2 and Level 4.
TABLE-US-00041 TABLE 40 Summary of HCV LOD/LOQ Data in the Roche
COBAS TaqMan HCV Assay Reported Frozen Sample Percent Percent Mean
Viral Viral Load Number Number Detected Number Quantitated Load
(LOG Level (LOG IU/mL) tested Detected (%) Quantitated (%) IU/mL) 3
2.25 20 20 100% 20 100% 1.89 4 2.14 20 20 100% 15 75% 1.58 5 1.85
20 20 100% 0 0% N/A 6 1.55 20 15 70% 1 5% 1.57 Frozen Sample
Percent Percent Reported Viral Load Number Number Detected Number
Quantitated Mean Viral Level (IU/mL) tested Detected (%)
Quantitated (%) Load (IU/mL) 3 178 20 20 100% 20 100% 84 4 142 20
20 100% 15 75% 41 5 72 20 20 100% 0 0% N/A 6 37 20 15 70% 1 5%
37
Conclusion
[0347] For the LOD/LOQ study, the diluted plasma samples yielded
slightly higher HCV viral load values than expected; however,
linear regression analysis yielded an R.sup.2 value of 0.9946,
indicating the diluted samples were acceptable for use with the
LOD/LOQ study (Table 39 and FIG. 47). Based on the Probit analysis
of the data from the plasma samples processed through the ViveST
devices, when the plasma sample with a HCV RNA concentration of 161
IU/ml (2.21 LOG IU/mL), was loaded on the ViveST devices of the
invention and stored for 7 days at ambient condition (RT), that
sample was quantitated with 95% probability (FIG. 48). Probit
values were recalculated to determine the limit of detection (LOD).
This required calculating the viral load values for the samples
that were detected but not quantitated by the AmpliLink software
(i.e., results <25 IU/mL).
5. Final Conclusions
[0348] The use of the ViveST devices of the invention with the
Roche COBAS TaqMan HCV Test (v2.0) for use with The High Pure
System demonstrated acceptable precision, reproducibility,
accuracy, and stability. The results indicate that after 7 days
storage at an ambient condition (RT) .about.0.55 LOG IU/mL
reduction in HCV concentration is observed for the samples stored
and processed through the ViveST devices of the invention as
compared to the frozen plasma. The concentration of HCV RNA
quantitated with 95% probability after 7 days was 161 IU/mL. The
results demonstrate the ViveST devices' utility for storing HCV
infectious samples for viral load testing.
Example 20
Concentration Study for the ViveST Devices of the Invention
1. Purpose
[0349] The purpose of this study was to determine if more than 1.0
mL of specimen (i.e., up to 2.0 mL) can be successfully loaded onto
the ViveST devices of the invention. All specimens, regardless of
load volume, were recovered in 1.0 mL to ascertain if sensitivity
is improved by concentrating biological specimens. This study
describes the results of specimen load volume experiments, as well
as results of analyzing `concentrated specimens` compared to
`non-concentrated specimens`.
2. Methodology
[0350] HIV-1 infectious plasma was loaded on/recovered from the
ViveST device of the invention. Recovered specimens were analyzed
as outlined in the Abbott REALTIME HIV-1 Assay package insert and
in accordance with the bioMONTR Research Method (RM-002.00
Quantitation of HIV-1 RNA Using the Abbott REALTIME HIV-1
Assay).
3. Experimental Design
3.1 Specimen Loading Volume
[0351] To assess the maximum volume of plasma that can be
successfully loaded onto the ViveST devices of the invention with
the polyolefin matrix, HIV-1 infectious plasma (1.0 mL, 1.5 mL and
2.0 mL) was pipetted onto the top of each polyolefin matrix of the
individually labeled ViveST device. As described below, pictures
were taken to document the results.
[0352] Conclusion: 1.0 mL of plasma was loaded onto the polyolefin
matrix of the ViveST devices of the invention and was completely
absorbed at the time of loading. Additonal volume, up to 1.5 mL,
was loaded but was not completely absorbed until approximately 30
minutes after loading. Any volume above 1.5 mL was not appear to be
absorbed by the matrix. This excess volume appeard to dry on the
interior surface of the cap and could not be recovered for
analysis.
3.2 Abbott REALTIME HIV-1
[0353] To assess concentration of HIV-1 infectious plasma using the
ViveST devices of the invention, an HIV-1 infectious plasma sample
at a concentration of .about.2.08 LOG c/mL (.about.120 c/mL) was
analyzed. 10 replicates at 1 mL and 10 replicates at 1.5 mL each
were pipetted onto the top of each polyolefin matrix of each
individually labeled ViveST device.
[0354] The loaded matrixes were dried overnight in a laminar flow
hood at ambient temperature. Devices were capped and stored 4 days
at ambient temperature prior to recovery. All specimens were
recovered using 1 mL molecular grade water and analyzed according
to the Abbott REALTIME HIV-1 package insert (0.5 mL application).
The results are provided in Table 41 and FIG. 53.
TABLE-US-00042 TABLE 41 Individual Results of HIV-1 Concentration
Study using the Abbott REALTIME HIV-1 Assay Results Results Mean
Mean LOG Specimen ID c/mL LOG c/mL c/mL c/mL Std Dev 1 mL HIV 41
1.61 48 1.60 0.28 1 mL HIV 12 1.07 1 mL HIV 51 1.71 1 mL HIV 116
2.07 1 mL HIV 54 1.73 1 mL HIV 69 1.84 1 mL HIV 34 1.53 1 mL HIV 30
1.47 1 mL HIV 49 1.69 1 mL HIV 20 1.31 1.5 mL HIV 147 2.17 142 2.14
0.12 1.5 mL HIV 146 2.17 1.5 mL HIV 112 2.05 1.5 mL HIV 132 2.12
1.5 mL HIV 120 2.08 1.5 mL HIV 142 2.15 1.5 mL HIV 85 1.93 1.5 mL
HIV 154 2.19 1.5 mL HIV 250 2.4 1.5 mL HIV 136 2.13
[0355] Conclusion: An average value of 2.14 LOG c/mL was obtained
when 1.5 mL of a low titer HIV-1 infectious plasma sample was
loaded on the ViveST devices of the invention and recovered using
1.0 mL of molecular grade water compared to an average value of 1.6
LOG c/mL when 1 mL was loaded and recovered using 1.0 mL molecular
grade water. These results indicate that the ViveST devices of the
invention may be used to concentrate virus in plasma specimens.
4. Final Conclusions
[0356] Up to 1.5 mL of plasma can be successfully loaded on the
polyolefin matrix of the ViveST devices of the invention. Volumes
in excess of 1.0 mL were not immediately absorbed but can be fully
absorbed into the matrix after .about.30 minutes. Viral targets
were concentrated using the ViveST devices of the invention by
recovering a volume less than that loaded. However, there was not a
direct proportional relationship between the results obtained with
1 mL input compared to 1.5 mL input indicating that target
concentration with the ViveST devices of the invention could have a
more meaningful application for qualitative assays
(positive/negative tests).
Example 21
Low Titer HCV Study
1. Purpose
[0357] The purpose of this study was to evaluate performance of the
ViveST devices of the invention for storage of low titer HCV
infectious plasma. This study describes the results of the low
titer HCV study.
2. Methodology
[0358] All testing on the Abbott REALTIME HCV assay was performed
according to the FDA approved protocol (0.9 mL) with no
modifications. 1 mL HCV infectious plasma was loaded onto the
ViveST devices, dried, stored for 3, 4, 5, or 7 days at an ambient
temperature and recovered in 1 mL molecular grade water. HCV viral
load results of frozen samples were compared to the samples stored
and processed through the ViveST devices.
3. Experimental Design
[0359] A panel of low titer HCV infectious plasma samples (HCV Type
1b) was purchased from Qnostics. Material was shipped on dry ice
and stored at -80.degree. C. pending analysis. Qnostics provided
the test results: 1.76 LOG IU/mL when tested against the WHO
2.sup.nd International Standard; 2.14 LOG IU/mL when tested against
the WHO 4.sup.th International Standard; and assigned value of 100
IU/mL.
[0360] To confirm the viral load of the purchased material, 45
samples were thawed and analyzed without being processed through
the ViveST devices (i.e., frozen samples). To evaluate the
performance of samples stored and processed through the ViveST
devices of the invention, 180 samples were thawed, loaded onto the
ViveST devices (1 mL each), dried, and stored at ambient
conditions. 45 samples were recovered from the ViveST devices using
1 mL molecular grade water after storage for 3 days, 4 days, 5
days, and 7 days. Frozen samples and all recovered samples were
analyzed in the Abbott REALTIME HCV assay in accordance with the
package insert and the bioMONTR Research Method (RM-003.00
Quantitation of HCV RNA Using the Abbott REALTIME HCV Assay).
4. Results
[0361] A summary of the Abbott REALTIME HCV viral load results for
the frozen samples and samples stored and processed through the
ViveST devices of the invention is provided in Table 42 (LOG IU/mL)
and Table 43 (IU/mL) below.
[0362] The average concentration of the Qnostics panel samples
based on testing 45 frozen samples was 1.80 LOG IU/mL (69 IU/mL)
with a range of 1.56-2.20 LOG IU/mL (37-158 IU/mL). The average
viral load is below the Qnostics' assigned value of 100 IU/mL. 100%
of the samples stored and processed through the ViveST devices of
the invention were detected with an average viral load of: 1.35 LOG
IU/mL (23 IU/mL) when stored for 3 days at ambient temperature
(n=45); 1.29 LOG IU/mL (21 IU/mL) when stored for 4 days at ambient
temperature (n=45); 1.27 LOG IU/mL (20 IU/mL) when stored for 5
days at ambient temperature (n=45); and 1.26 LOG IU/mL (19 IU/mL)
when stored for 7 days at ambient temperature (n=45).
[0363] The Standard Deviations across all assays were: 0.17 LOG
IU/mL for frozen samples (n=45); 0.12 LOG IU/mL when stored for 3
days at ambient temperature (n=45); 0.17 LOG IU/mL when stored for
4 days at ambient temperature (n=45); 0.19 LOG IU/mL when stored
for 5 days at ambient temperature (n=45); and 0.13 LOG IU/mL when
stored for 7 days at ambient temperature (n=45).
[0364] The average reduction in viral load for the samples stored
and processed through the ViveST devices of the invention, when
compared to the frozen plasma, was: 0.45 LOG IU/mL when stored for
3 days at ambient temperature (n=45); 0.51 LOG IU/mL when stored
for 4 days at ambient temperature (n=45); 0.53 LOG IU/mL when
stored for 5 days at ambient temperature (n=45); and 0.54 LOG IU/mL
when stored for 7 days at ambient temperature (n=45).
[0365] As shown in FIG. 54, a scatter plot indicates no noticeable
trends other than all the samples stored and processed through the
ViveST devices yield lower results than the frozen plasma. While
the average for the frozen plasma (n=45) was 1.80 LOG IU/mL, all
the samples stored and processed through the ViveST devices (n=180)
yielded results between 0.71 LOG IU/mL-1.77 LOG IU/mL. Results
reported as <1.08 LOG IU/mL (<12 IU/mL) by the Abbott
REALTIME HCV data analysis software were manually calculated using
a stored HCV calibration curve.
TABLE-US-00043 TABLE 42 Summary of Low Copy HCV Study (LOG IU/mL)
Replicate of Qnostics Abbott RealTime HCV (LOG IU/mL) HCV panel
Samples FROZEN Day 3 Day 4 Day 5 Day 7 1 1.82 1.44 1.28 1.53 1.21 2
1.89 1.18 1.36 0.93 1.39 3 1.75 1.33 1.15 1.47 1.09 4 2.11 1.55
1.17 1.03 1.31 5 1.76 1.40 1.08 1.29 1.53 6 1.89 1.40 1.29 1.26
1.34 7 2.00 1.39 0.91 1.19 1.26 8 2.12 1.33 1.31 0.88 1.32 9 1.75
1.43 1.77 1.47 1.42 10 1.77 1.32 1.11 1.34 1.31 11 1.79 1.35 1.31
1.31 1.42 12 1.81 1.27 1.48 1.35 1.28 13 1.64 1.18 1.05 1.36 1.38
14 1.88 1.22 1.37 1.15 1.06 15 1.81 1.41 1.21 1.15 1.05 16 2.17
1.42 1.52 1.25 1.25 17 1.69 1.55 1.19 1.36 1.39 18 1.83 1.14 1.41
1.48 1.33 19 1.71 1.31 1.44 1.06 1.29 20 2.20 1.38 1.22 1.17 1.33
21 1.70 1.27 1.44 1.21 1.23 22 2.00 1.53 1.36 1.41 0.88 23 1.64
1.55 1.34 0.71 1.18 24 1.80 1.31 1.15 1.25 1.23 25 1.68 1.28 1.43
1.42 1.17 26 1.70 1.37 1.34 1.14 1.03 27 2.19 1.48 1.15 1.46 1.3 28
1.68 1.37 1.10 0.91 1.2 29 1.85 1.37 1.10 1.13 1.13 30 1.87 1.43
1.49 1.07 1.43 31 1.89 1.39 1.00 1.39 1.32 32 1.78 1.30 1.34 1.52
1.37 33 1.73 1.54 1.18 1.3 1.07 34 1.56 1.30 1.38 1.3 1.26 35 1.59
1.36 1.46 1.71 1.15 36 1.73 1.27 1.27 1.47 1.11 37 1.69 1.44 1.25
1.21 1.21 38 1.58 1.46 1.51 1.55 1.35 39 1.61 1.20 1.20 1.37 1.4 40
1.69 0.98 1.19 1.3 1.15 41 1.60 1.29 1.66 1.35 1.06 42 1.77 1.21
1.36 1.38 1.33 43 2.08 1.39 1.15 1.45 1.24 44 1.71 1.55 1.42 1.09
1.35 45 1.69 1.17 1.37 1.39 1.47 Average (n = 45) 1.80 1.35 1.29
1.27 1.26 Std Dev 0.17 0.12 0.17 0.19 0.13 95% CI 0.05 0.04 0.05
0.05 0.04
TABLE-US-00044 TABLE 43 Summary of Low Copy HCV Study (IU/mL)
Replicate of Qnostics Abbott RealTime HCV (IU/mL) HCV panel Samples
FROZEN Day 3 Day 4 Day 5 Day 7 1 66 27 19 34 16 2 78 15 23 9 24 3
57 22 14 30 12 4 129 35 15 11 20 5 58 25 12 20 34 6 77 25 20 18 22
7 100 24 8 15 18 8 133 21 20 8 21 9 57 27 58 29 27 10 53 21 13 22
20 11 62 23 20 20 26 12 65 19 30 22 19 13 44 15 11 23 24 14 76 17
21 14 11 15 64 26 16 14 11 16 149 26 33 18 18 17 49 36 16 23 24 18
67 14 26 30 22 19 51 20 27 11 19 20 158 24 17 13 21 21 51 19 27 16
17 22 100 34 23 26 8 23 44 35 22 5 15 24 64 20 14 18 17 25 48 19 27
27 15 26 50 23 22 14 11 27 153 30 14 29 20 28 48 23 13 8 16 29 71
24 13 14 14 30 66 27 31 12 27 31 78 24 10 25 21 32 60 20 22 33 23
33 53 34 15 20 12 34 37 20 24 20 18 35 39 23 29 16 14 36 53 19 19
30 13 37 49 27 18 16 16 38 38 29 32 35 22 39 41 16 16 24 25 40 49
10 15 20 14 41 40 19 45 23 11 42 58 16 23 22 21 43 106 24 14 28 17
44 51 35 27 12 22 45 49 15 23 24 26 Average (n = 45) 69 23 21 20 19
Std Dev 32 6 9 8 5 95% CI 9 2 3 2 2
5. Final Conclusions
[0366] Frozen plasma samples (n=180) with an average viral load of
1.80 LOG IU/mL (69 IU/mL) were stored on the ViveST devices of the
invention for up to 7 days. Upon recovery, 100% of these samples
were detected using the Abbott's REALTIME HCV assay. While there
was some reduction in viral load, the recovery was very
reproducible regardless of storage time. These data support the use
of a correction factor of 0.5 LOG IU/mL to normalize/align the
viral load with values that would be obtained from frozen plasma
for the samples stored and processed through the ViveST devices of
the invention.
Example 22
Validation of the Samples Processed through the ViveST Devices for
Use with the Abbott REALTIME HBV Assay
1. Purpose
[0367] The study purpose was to validate the samples processed
through the ViveST devices of the invention for use in the Abbott
REALTIME HBV assay. This study describes the results of: precision
and accuracy studies; linearity (analytical measurement range);
stability (7 days); accuracy as compared to the frozen plasma; and
limit of detection (LOD)/limit of quantitation (LOQ).
2. Methodology
[0368] The Abbott REALTIME HBV assay is an in vitro polymerase
chain reaction (PCR) based assay for the quantitation of Hepatitis
B Virus (HBV) DNA in human plasma (EDTA) from chronically
HBV-infected individuals. The process is based on two major steps:
a) extraction of viral DNA from plasma samples; and b)
amplification with concurrent detection of viral DNA.
3. Experimental Design
[0369] All testing on the Abbott REALTIME HBV assay was performed
according to the FDA approved protocol (0.5 mL) with no
modifications, and in accordance with the bioMONTR Research Method
(RM 008.00, Quantitation of HBV DNA Using the Abbott RealtTime HBV
Assay). The Abbott HBV assay (0.5 mL protocol) requires 0.7-1.2 mL
sample, therefore, 1.0 mL sample was loaded on/recovered from the
ViveST devices of the invention to ensure adequate sample volume.
All loaded ViveST devices were stored at an ambient temperature
(RT). The performance of samples stored and processed through the
ViveST devices of the invention in this assay was evaluated for
precision/accuracy, analytical measurement range, stability, and
limit of detection (LOD)/limit of quantitation (LOQ).
4.1 Precision (Inter and Intra-Assay Precision)
[0370] To assess inter- and intra-assay precision, HBV infectious
plasma samples with varying viral load values (low, mid, and high
viral load) were stored in triplicate on the ViveST devices,
recovered, and tested with the Abbott REALTIME HBV assay on
different days (n=27). A summary of the results is provided in
Table 44 below.
TABLE-US-00045 TABLE 44 Summary of Intra-Assay and Inter-assay
Precision (Mean Values) Abbott REALTIME HBV_Intra-assay and
Inter-assay Precision Intra-Assay Precision Inter-Assay Precision
Concentration Low Medium High Run # 1 2 3 1 2 3 1 2 3 Days Stored 1
4 7 1 4 7 1 4 7 Low Medium High Replicates 3 3 3 3 3 3 3 3 3 9 9 9
Mean (log IU/mL) 3.61 3.57 3.66 4.74 4.70 4.83 5.81 5.82 5.84 3.61
4.76 5.82 Standard Deviation 0.04 0.05 0.06 0.02 0.13 0.10 0.02
0.04 0.03 0.06 0.10 0.03 95% Confidence 0.04 0.05 0.07 0.02 0.14
0.11 0.03 0.04 0.03 0.04 0.06 0.02 Interval
Conclusion: The inter-assay and intra-assay standard deviations
(SDs) achieved at mean concentrations of .about.3.6, .about.4.7 and
.about.5.8 LOG IU/mL are <0.13 log IU/mL indicating robust
reproducibility. The coefficient of variation (% CV) at a 95%
confidence interval for inter-assay precision was <0.06% for all
time points for all sample concentrations. The coefficient of
variation (% CV) at a 95% confidence level for intra-assay
precision was <0.14% for all time points for all sample
concentrations.
4.2 Analytical Measurement Range and Accuracy
[0371] For testing analytical measurement range, a high titer HBV
infectious plasma sample (.about.7 log IU/mL) was serially diluted
in normal human plasma (7 levels). Each level was loaded onto the
ViveST devices in triplicate, stored for 7 days, recovered, and
tested on a single run (n=21). For accuracy compared to frozen
plasma, identical serial dilutions were frozen (in triplicate),
thawed, and analyzed (N=21). Results are provided in Table 45 and
FIG. 55.
TABLE-US-00046 TABLE 45 HBV Linearity Using the Abbott REALTIME HBV
Assay ViveST Device Processed Sample versus Frozen Plasma_Abbott
REALTIME HBV (LOG IU/mL) Actual Average Results Results ViveST
Difference Sample Frozen Frozen ViveST Results Standard Frozen Vs
ID Samples Samples Results Average Deviation ViveST Level 7 1.14
0.92 1.07 1.07 0.05 0.15 0.75 1.03 0.87 1.12 Level 6 2.15 2.09 2.15
2.09 0.07 0.00 1.97 2.02 2.15 2.09 Level 5 2.81 2.86 2.83 2.82 0.07
-0.04 2.90 2.74 2.86 2.88 Level 4 3.79 3.80 3.82 3.76 0.05 -0.04
3.77 3.74 3.85 3.73 Level 3 4.93 4.87 4.83 4.84 0.04 -0.02 4.82
4.81 4.85 4.89 Level 2 6.00 5.93 5.90 5.89 0.01 -0.04 5.85 5.88
5.93 5.88 Level 1 7.00 6.97 6.96 6.97 0.03 -0.01 6.90 7.00 7.02
6.94 Minimum Loss 0.15 Maximum Loss -0.04 Average Loss (7 day
storage) 0.00
[0372] Conclusion: This study confirmed exceptional sample
correlation across a range of .about.1 to .about.7 LOG (samples
stored and processed through the ViveST devices of the invention as
compared to the frozen plasma) with linear regression analysis
yielding an R.sup.2 value of 0.99706. On average, sample recovered
from the ViveST devices of the invention after being stored at an
ambient condition (RT) for 7 days yielded equivalent viral load
values as compared to frozen plasma.
4.3 Stability
[0373] To assess stability, HBV infectious plasma samples with
varying viral load values (low, mid, and high viral load) were
analyzed on the Abbott REALTIME HBV assay after being stored at an
ambient condition (RT) on the ViveST devices of the invention for
1, 4, 7, 14, 30, and 60 days. For accuracy as compared to the
frozen plasma, identical serial dilutions were frozen (in
triplicate), thawed, and analyzed (N=21). Results are provided in
Table 46, FIG. 56 and FIG. 57.
TABLE-US-00047 TABLE 46 Results Summary for the HBV 60-Day
Stability Studies at Ambient Storage Conditions using the Abbott
REALTIME HBV Assay Target HBV titre Frozen Mean Results (LOG
IU/mL): Ambient Storage (Days) Level (LOG IU/mL) (LOG IU/mL) 1 4 7
14 30 60 1 5.97 5.81 5.81 5.82 5.84 5.91 5.83 5.83 2 4.97 4.71 4.74
4.70 4.78 4.83 4.71 4.77 3 3.97 3.71 3.61 3.57 3.66 3.74 3.71
3.70
[0374] Conclusion: For samples stored on the ViveST devices of the
invention over a 60-day period at an ambient conditions (RT) there
was no reduction of HBV DNA when compared to the frozen plasma
(Table 46 and FIG. 57). The Standard Deviation across all
levels/all test points ranged from 0.02 to 0.13. A linear fit
(R.sup.2>0.99) was retained over the course of the 60-day study
as indicated by linear regression analysis across all time points
(FIG. 56).
4.4 Limit of Detection (LOD)/Limit of Quantitation (LOQ)
[0375] For determination of the LOD/LOQ, HBV infectious plasma was
diluted in HBV negative human plasma to yield dilutions of
approximately 1.5 to 50 IU/mL. To confirm the HBV DNA
concentration, the diluted samples were analyzed and linear
regression analysis was performed. 15 replicates of each
concentration were then loaded onto the ViveST devices of the
invention and stored for 7 days at an ambient condition (RT). After
recovery, samples were tested using a single lot of extraction and
amplification reagents. Probit analysis was performed to determine
the 95% hit rate.
TABLE-US-00048 TABLE 47 HBV LOD/LOQ Studies for the Frozen Plasma
Samples Not Processed through the ViveST Devices using the Abbott
REALTIME HBV Assay Frozen Plasma: Determination HBV LOD results
Target Target Achieved Average Achieved Average titre titre (log
HBV titre HBV titre HBV titre HBV titer Level (IU/mL) IU/mL) Sample
ID (log IU/mL) (log IU/mL) (IU/mL) (IU/mL) 6 1.5 0.18 frozen L6
1.01 0.67 10 6 0.32 2 5 3 0.48 frozen L5 0.76 0.79 6 7 0.82 7 4 6
0.78 frozen L4 1.1 0.99 13 11 0.88 8 3 12 1.08 frozen L3 1.47 1.17
30 19 0.86 7 2 25 1.40 frozen L2 1.77 1.68 58 49 1.59 39 1 50 1.70
frozen L1 1.85 1.85 70 70
TABLE-US-00049 TABLE 48 Summary of LOD/LOQ Data in the Abbott
REALTIME HBV Assay Mean Frozen Sample Viral Load Viral Load Number
Number Percent (LOG Level (LOG IU/mL) tested Detected Detected (%)
IU/mL) 6 0.67 15 14 93% 0.62* 5 0.79 15 15 100% 0.71* 4 0.99 15 15
100% 1.06* 3 1.17 15 15 100% 1.42 2 1.68 15 15 100% 1.72 1 1.85 15
15 100% 1.97 Frozen Sample Mean Viral Load Number Number Percent
Viral Load Level (IU/mL) tested Detected Detected (%) (IU/mL) 6 6
15 14 93% 4* 5 7 15 15 100% 6* 4 11 15 15 100% 12* 3 19 15 15 100%
27 2 49 15 15 100% 54 1 70 15 15 100% 95 *Results reported as
<1.00 LOG IU/mL (<10 IU/mL) by the Abbott REALTIME HBV data
analysis software were manually calculated using a stored HBV
calibration curve.
[0376] Conclusion: For the LOD/LOQ study, the diluted plasma
samples yielded slightly higher HBV viral load values than
expected; however, linear regression analysis yielded an R.sup.2
value of 0.9575, indicating the diluted samples were acceptable for
use with the LOD/LOQ study (Table 47 and FIG. 58). As summarized in
Table 48, 14 of 15 samples or 93% with an estimated viral load of 6
IU/mL were detected and yielded a manually calculated mean viral
load of 4 IU/mL. All samples (15 of 15) with an estimated viral
load of 7 IU/mL were detected and yielded a manually calculated
mean viral load of 6 IU/mL.
[0377] Probit analysis was performed on all the samples stored and
processed through the ViveST devices of the invention and analyzed
and quantitated by the Abbott REALTIME HBV data analysis software.
Based on this analysis, when the plasma sample with a HBV DNA
concentration of 13 IU/ml (1.10 LOG IU/mL) was loaded on the ViveST
devices of the invention and stored for 7 days at an ambient
condition (RT), that sample was quantitated with 95% probability
(FIG. 59). The Probit analysis was performed using only sample
values quantitated by the Abbott Software (i.e., >10 IU/mL).
5. Final Conclusions
[0378] The use of the samples stored and processed through the
ViveST devices of the invention with the Abbott REALTIME HBV assay
demonstrated acceptable precision, reproducibility, accuracy, and
stability. The results confirm that HBV infectious plasma stored on
the ViveST devices of the invention yields results comparable to
those obtained from the frozen plasma not processed through the
ViveST devices of the invention.
[0379] Other embodiments and uses are apparent to one skilled in
the art in light of the present disclosures. Those skilled in the
art will appreciate that numerous changes and modifications can be
made to the embodiments of the invention and that such changes and
modifications can be made without departing from the spirit of the
invention. It is, therefore, intended that the appended claims
cover all such equivalent variations as fall within the true spirit
and scope of the invention.
* * * * *